Compounds and pharmaceutical compositions for the treatment and prophylaxis of bacterial infections

ABSTRACT

Novel methods for the treatment and/or prophylaxis of diseases caused by tissue-adhering bacteria are disclosed. By interacting with periplasmic molecular chaperones it is achieved that the assembly of pili is prevented or inhibited and thereby the infectivity of the bacteria is diminished. Also disclosed are methods for screening for drugs as well as methods for the de novo design of such drugs, methods which rely on novel computer drug modelling methods involving an approximative calculation of binding free energy between macromolecules. Finally, novel pyranosides which are believed to be capable of interacting with periplasmic molecular chaperones are also disclosed.

This invention was made with US government support under grant numberR01AI29549 (S.J.H.) and training grant AI07172, both awarded by NIH. TheUS government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to methods for the treatment and/orprophylaxis of diseases caused by tissue-adhering pilus-forming bacteriaby interaction with the binding between pilus subunits and periplasmicchaperones. The invention further relates to methods foridentifying/designing substances capable of interacting with periplasmicchaperones and methods for identifying binding sites in periplasmicchaperones. Finally, the invention relates to novel substances capableof interacting with periplasmic chaperones as well as pharmaceuticalpreparations comprising substances capable of interacting withperiplasmic chaperones.

BACKGROUND OF THE INVENTION

Pathogenic Gram-negative bacteria cause a number of pathologicalconditions such as bacteraemia, bacteria-related diarrhoea, meningitisand (very commonly) urinary tract infections, i.a. pyelonephritis,cystitis, urethritis etc.

Urinary tract infections are one of the major causes of morbidity infemales. Despite the overall importance of urinary tract infections inwomen, there have been few efforts to apply novel strategies in order totreat and/or prevent these diseases. Commonly, conventional antibioticsare used to treat these infections, such as treatment with penicillins,cephalosporins, aminoglycosides, sulfonamides and tetracyclines; in thespecial case of urinary tract infections, urinary antiseptics such asnitrofurantoin and nalidixic acid are employed, too. However, emergingantibiotic resistance will in the future hamper the ability tosuccessfully treat urinary tract infections. Multiple antibioticresistance among these uropathogens is increasing. It has been estimatedthat the annual cost for evaluation and treatment of women with urinarytract infections exceeds one billion dollars. In addition, approximatelyone-fourth of the yearly 4 billion dollar cost attributed to nosocomialinfections is a consequence of urinary tract infections. Among thecausative agents of urinary tract infections, Escherichia coli clearlypredominates among Gram-negative bacteria.

Pathogenic gram negative bacterial notably Escherichia coli, Haemophilusinfluenzae, Salmonella enteriditis, Salmonella typhimurium, Bordetellapertussis, Yersinia pestis, Yersinia enterocolitica, Helicobacterpylori, and Klebsiella pneumoniae owe part of their infectability totheir ability to adhere to various epithelial tissues. Thus, e.g. E.coli adhere to the epithelial cells in the upper urinary tract in spiteof the flushing effect of unidirectional flow of urine from the kidneys.

As indicated above, the above mentioned bacteria are involved in avariety of diseases: Urinary tract infections (E. coli), acute diarrhoea(E. coli, Y. enterocolitica and Salmonella spp), meningitis (E. coli andH. influenzae), whooping cough (B. pertussis), plague (Y. pestis),pneumonia and other respiratory tract infections (K. pneumoniae, H.influenzae) and peptic ulcer (H. pylori).

The initiation and persistence of many bacterial infections such asthose described above is thought to require the presentation of adhesinson the surface of the microbe in accessible configurations which promotebinding events that dictate whether extracellular colonization,internalization or other cellular responses will occur. Adhesins areoften components of the long, thin, filamentous, heteropolymeric proteinappendages known as pili, fimbriae, or fibrillae (these three terms willbe used interchangeably herein) The bacterial attachment event is oftenthe result of a stereo-chemical fit between an adhesin frequentlylocated at the pilus tip and specific receptor architectures on hostcells, often comprising carbohydrate structures in membrane associatedglycoconjugates.

Uropathogenic strains of E. coli express P and type 1 pili that bind toreceptors present in uroepithelial cells. The adhesin present at the tipof the P pilus, PapG (pilus associated polypeptide G), binds to theGalα(1-4)Gal moiety present in the globoseries of glycolipids, while thetype 1 adhesin, FimH, binds D-mannose present in glycolipids andglycoproteins. Adhesive P pili are virulence determinants associatedwith pyelonephritic strains of E. coli whereas type 1 pili appear to bemore common in E. coli causing cystitis. At least eleven genes areinvolved in the biosynthesis and expression of functional P pili; theDNA sequence of the entire pap gene cluster has been determined. P piliare composite heteropolymeric fibers consisting of flexible adhesivefibrillae joined end to end to pilus rods. The pilus rod is composed ofrepeating PapA protein subunits arranged in a right handed helicalcylinder. Tip fibrillae which extend from the distal ends of each pilusrod were found to be composed mostly of repeating subunits of PapEarranged in an open helical conformation. The PapG adhesin was localizedto the distal ends of the tip fibrillae, a location which is assumed tomaximize its ability to recognize glycolipid receptors on eukaryoticcells. Two minor pilus components, PapF and PapK, are specializedadaptor proteins found in the tip fibrillum. PapF links the adhesinmoiety to the fibrillum while PapK joins the fibrillum to the pilus rod.The composite architecture of the P pilus fibre reveals the strategyused by uropathogenic E. coli to present the PapG adhesin to eukaryoticreceptors. The rigid PapA rod extends the adhesin away from interferencecaused by LPS and other components at the bacterial cell surface whilethe flexible fibrillum allows PapG steric freedom to recognize and bindto the digalactoside moiety on the uroepithelium. With a few exceptions,the structural organization of type 1 pili is very similar to thatdescribed for P pili. In type 1 pili, the mannose binding fibrillar tipadhesin is known as FimH.

The assembly of virulence-associated pili in Gram-negative pathogensrequires the function of periplasmic chaperones. Molecular chaperonesare vital components of all living cells, prokaryotic and eukaryotic.Chaperones serve a variety of cellular functions including folding,import and export of proteins in various cellular compartments (Gethingand Sambrook, 1992). Thus, a periplasmic chaperone is a molecularchaperone which exerts its action in the periplasmic space in bacteria.

PapD and FimC are the periplasmic chaperones that mediate the assemblyof P and type 1 pili, respectively. Detailed structural analyses haverevealed that PapD is the prototype member of a conserved family ofperiplasmic chaperones in Gram-negative bacteria. These chaperones havea function which is part of a general strategy used by bacteria to capand partition interactive subunits imported into the periplasmic spaceinto assembly competent complexes, making non-productive interactionsunfavourable. Determination of the three-dimensional structure of PapDrevealed that it consists of two immunoglobulin-like domains oriented ina boomerang shape such that a cleft is formed. PapD binds to each of thepilus subunit types as they emerge from the cytoplasmic membrane andescorts them in assembly-competent, native-like conformations from thecytoplasmic membrane to outer membrane assembly sites comprised of PapC.PapC has been termed a molecular usher since it receiveschaperone-subunit complexes and incorporates, or ushers, the subunitsfrom the chaperone complex into the growing pilus in a defined order.

With the exception of the type IV class of pili, all other geneticallywell characterized pilus systems in Gram-negative prokaryotes contain agene analogous to PapD (Normark et al., 1986; Hultgren et al. 1991); cf.also table A. FanE, faeE, sfaE, ClpE and f17-D have been sequenced(Lintermans, 1990; Schmoll et al., 1990; Bertin Y et al., 1993; Bakkeret al., 1991) and encode pilus chaperones required for the assembly ofK99, K88, S and F17 pili, respectively, in E. coli. The assembly ofKlebsiella pneumoniae type 3 pili and Haemophilus influenzae type b pilirequires the mrkb and hifB gene products, respectively (Gerlach et al.,1989,; Allen et al., 1991). The structure-function relationships of allof these chaperones have been analyzed using their amino acid sequencesand information from the crystal structure of PapD (Anders Holmgren etal., 1992). The results have provided insight into the molecularintricacies that have been evolutionarily conserved in this class ofproteins and suggested significant structural similarities toimmunoglobulins.

PapD is thus the prototype member of a family of periplasmic chaperoneproteins which are necessary for the correct supramolocular assembly ofbacterial pili. Chaperones such as PapD in E. coli are required to bindthe above-mentioned pilus proteins imported into the periplasmic space,partition them into assembly competent complexes and preventnon-productive aggregation of the subunits in the periplasm (Dodson etal. 1993).

In the absence of an interaction with the chaperone, pilus subunitsaggregate and are proteolytically degraded (Kuehn, Normark, andHultgren, 1991). It has recently been discovered (Strauch, Johnson andBeckwith, 1989) that the DegP protease is greatly responsible for thedegradation of pilin subunits in the absence of the chaperone. Thisdiscovery has allowed the elucidation of the fate of pilus subunitsexpressed in the presence or absence of the chaperone using monospecificantisera in western blots of cytoplasmic membrane, outer membrane andperiplasmic proteins prepared according to standard procedures.Expression of papG or papA in the degP41 strain (a DegP⁻ E. coli strain)in the absence of a chaperone was toxic to the bacteria due to theaccumulation of these proteins in the cytoplasmic membrane suggestingthat the chaperone was required for subunit import into the periplasmicspace. The severity of the growth defect was related to the level ofexpression of the pilin subunit, and was generally more dramatic withPapG than PapA. Co-expression of PapD under the control of the induciblearabinose promoter rescued the growth defect associated with subunitexpression in the degP41 strain and allowed PapG to be imported into theperiplasm.

Up to this time, little has been elucidated about the molecularrecognition motifs of chaperones. Cytoplasmic chaperones such as SecB,GroEL and DNaK bind to a diverse group of unfolded target proteins in asequence independent manner (Gething and Sambrook, 1992). Recently, ithas been suggested that DNaK binds mainly via the target's peptidebackbone (Landry et al., 1992) and that GroEL may rely more onside-chain hydrophobicity and the ability of the target sequence to forman amphipathic α-helix (Landry and Gierasch, 1991). PapD differs,however, in that it seems to bind its target proteins in foldedconformations (Kuehn et al., 1991).

The three-dimensional structure of PapD has previously been solved(Holmgren and Bränden, 1989). This has shown PapD to consist of twoglobular domains positioned such that the overall shape of the moleculeresembles a boomerang with a cleft between the two domains. Each domainis a β-barrel structure formed by two antiparallel β-pleated sheets,packed tightly together to form a hydrophobic core, with a topologysimilar to that of an immunoglobulin fold. The N-terminal domain of PapDmost resembles an Ig variable domain whilst the C-terminal domain ofPapD resembles CD4 (Wang et al., 1990, Ryu et al., 1990) and the humangrowth hormone receptor (de Vos et al., 1992).

A structural alignment between PapD and several periplasmic chaperonespredicted to have a similar immunoglobulin-like structure has identifiedinvariant, highly conserved and variable residues within this proteinfamily (Holmgren et al., 1992). Most conserved residues seem toparticipate in maintaining the overall structure and orientation of thedomains towards one another. However, two conserved residues, Arg-8 andLys-112 are surface exposed and oriented towards the cleft between thedomains. Site-directed mutagenesis of the Arg-8 amino acid has shownthat it form at least part of the pilus subunit binding pocket (Holmgrenet al., 1992; Kuehn et al., 1993).

From a sequence analysis of a number of the above-mentioned pilussubunit proteins, it has been observed that they possess a number ofcommon features including homologies at the C termini (see also example2). It is thought that these similarities in sequence may be responsiblefor some function common to all the pilus proteins, such as binding totheir periplasmic chaperone. Indeed, the C-terminal region of the P-piliadhesin PapG has already been shown to be important in the in vivobinding to PapD (Hultgren et al., 1989). Table A lists 16 periplasmicproteins, all involved in assembly of cell surface structures inpathogenic bacteria and all with significant homology with PapD.

TABLE A Chap- Structure Organism erone Assembled Reference E. coli PapDP pili Lund et al., 1987 E. coli* FimC Type 1 pili Klemm et al., 1992 E.coli Sfae S pili Schmoll et al., 1990 E. coli FaeE K88 pili Bakker etal., 1991 E. coli FanE K99 pili Bakker et al., 1991 E. coli CS3-1 CS3pili Jalajakumari et al., 1989 E. coli F17D F17 pili Lintermaris et al.,1990 E. coli ClpE CS31 A Bertin et al., 1993 E. coli EcpD not identifiedRaina etal., 1993 E. coli CssC Antigen CS6 E. coli NfaE Nonfimbrialadhesin 1 E. coli AggD Aggregative Adherence Fimbria 1 K. pneumaniaeMrkB Type 3 pili Allen et al., 1991 B. pertussis FimB Type 2 & 3 piliLocht et al., 1992; Willems et al. 1992 S. enteriditis SefB unknown piliClouthier et al., 1993 S. typhimurium PefD PEF H. influenzae HifBunknown pili Smith et al., 1993 Y. enterocolitica MyfB Myf fibrillaeIriate et al., 1993 Y. pestis PsaB pH6 antigen Lindler et al., 1992 Y.pestis Caf1M F1 envelope Galyov et al., 1991 antigen P. mirabilis MrpDMR/P Fimbriae ? YehC ? *This chaperone is present in allEnterobacteriaceae, since all members of this family produce type 1pili.

In summary, the three-dimensional structure of PapD as well as thefunction of PapD and other periplasmic chaperones are known, whereas theexact motif of binding between PapD and the pilus subunits has beenunknown until now.

DISCLOSURE OF THE INVENTION

According to the present invention, it has been realized that theabove-mentioned characteristics of PapD and other related chaperonesmake them interesting targets for drugs designed primarily to reduce thepathogenicity of bacteria which adhere by means of pili; for example, adrug which blocks the binding between a chaperone and the pilus-subunits(thereby interfering with the assembly of the intact pilus) willinterfere with the formation of intact pili, thereby reducing bacterialcapacity to adhere to host epithelium.

In order to design such a drug it is of great value that the motif ofbinding between the chaperone and the pilus protein(s) is known indetail, in order to develop the method to effectively identify compoundscapable of blocking this binding.

An aspect of the current invention relates to a method for the treatmentand/or prophylaxis of diseases caused by tissue-adhering pilus-formingbacteria, comprising preventing, inhibiting or enhancing binding betweenat least one type of pilus subunit and at least one type of molecularchaperone in the pilus-forming bacteria, which molecular chaperone bindspilus subunits during transport of these pilus subunits through theperiplasmic space and/or during the process of assembly of the intactpilus.

As used herein, the term “pilus”, “fimbria”, or “fibrilla”, relates tofibrillar heteropolymeric structures embedded in the outer membrane ofmany tissue-adhering pathogenic bacteria, notably the pathogenic gramnegative bacteria. In the present specification the terms pilus,fibrillum and fimbria will be used interchangeably. A pilus is, asexplained above, composed of a number of “pilus subunits”, whichconstitute distinct functional parts of the intact pilus. A veryimportant pilus subunit is the “adhesin”, the pilus subunit which isresponsible for the tissue-binding capacity of the bacterium.

By the term “molecular chaperone” is meant a molecule which in livingcells has the responsibility of binding to peptides in order to maturethe peptides in a number of ways. Many molecular chaperones are involvedin the process of folding of peptides into their native conformationwhereas other molecular chaperones are involved in the process of exportout of or import into the cell of peptides. Specialized molecularchaperones are “periplasmic chaperones”, which are bacterial molecularchaperones exerting their main actions in the “periplasmic space” (thespace between the inner and outer bacterial membrane). Periplasmicchaperones are involved in the process of correct assembly of intactpili. When used herein, the simple term “chaperone” designates amolecular, periplasmic chaperone if nothing else is indicated.

When using the phrase “one type of” is meant that the pilus subunit orthe chaperone in question is of one distinct species. However,especially the fact that there is extensive homology between differentspecies of periplasmic molecular chaperones renders it likely that theinterference with one type of chaperone using e.g. a compound will makeit possible to also use the compound in interference with otherchaperones.

The phrase “preventing, inhibiting or enhancing binding between pilussubunits and at least one molecular chaperone in the pilus-formingbacteria” indicates that the normal interaction between a chaperone andits natural ligand, i.e. the pilus subunit, is being affected either bybeing completely or substantially completely prevented, or by beinginhibited, or expressed in another manner, reduced to a such an extentthat the binding of pilus subunits to the chaperone is measurably lowerthan is the case when the chaperone is interacting with the pilussubunit at conditions which are substantially identical (with regard topH, concentration of ions and other molecules) to the native conditionsin the periplasmic space. Similarly, the enhancement of binding betweenthe chaperone and the pilus subunit should be such that the binding ofpilus subunits to the chaperone is measurably higher than is the casewhen the chaperone is interacting with the pilus subunit at conditionswhich are substantially identical (with regard to pH, concentration ofions and other molecules) to the native conditions in the peri-plasmicspace. Measurement of the degree of binding can be determined in vitroby methods known to the person skilled in the art (microcalormetry,radioimmunoassays, enzyme based immuno assays, etc).

It should, on the basis of the above, be clear that prevention orinhibition of the normal interaction between a pilus subunit and achaperone should have a substantially limiting effect on pilus assembly.However, an enhancement of the binding between pilus subunits andchaperones may also prove to be devastating to a bacterium. As willappear from example 2, different pilus subunits bind to PapD withdifferent affinities and affecting this narrowly balanced system mayalso cause a limitation on the rate and efficiency of pilus assembly.

It is believed that even modest changes in the binding between pilussubunits and chaperones can have dramatic impact on the efficiency ofpilus assembly, and thus on the ability of the bacteria to adhere. Forexample, if the change in the binding between a chaperone and one pilussubunit is such that the normal order of affinities between thechaperones and the pilus subunits which normally bind thereto isaltered, then the normal assembly of the pilus should be disturbed,since the order of assembly of the pilus may be dependent i.a. on theaffinities between the pilus subunits and the chaperone: The pilussubunits with the highest affinities to the chaperone may beincorporated before other pilus subunits with lesser affinities.

Thus, prevention, inhibition or enhancement of binding between pilussubunits and a periplasmic molecular chaperone have the effect ofimpairing pilus assembly, whereby the infectivity of the microorganismnormally expressing the pili is reduced.

Prevention, inhibition or enhancement of the binding between pilussubunits can be accomplished in a number of ways. A preferred methodaccording to the invention of treatment and/or prophylaxis of diseasescaused by tissue-adhering pilus-forming bacteria is to administer aneffective amount of a substance to a subject in need thereof, thesubstance being capable of interacting with at least one type ofmolecular chaperone which binds pilus subunits during transport of thesepilus subunits through the periplasmic space and/or during the processof assembly of the intact pilus, in such a manner that binding of pilussubunits to the molecular chaperone is prevented, inhibited or enhanced.

The substance can be any compound which has one of the above mentionedeffects on the interaction between chaperones and pilus subunits andthereby on the assembly of the pilus. Especially interesting substancesare those which are likely to interact with the pilus subunit bindingpart of the chaperone, but interaction with other sites in thechaperones may also cause prevention, inhibition or enhancement of thebinding between pilus subunits and the chaperone. This can be an effectof direct steric blocking of the normal binding between the subunit andthe chaperone, but it may also be an effect of a conformational changein the chaperone. A method of identifying substances to be used in themethod of the invention is disclosed below.

The interaction between the substance and the chaperone may be acovalent as well as a non-covalent binding to the chaperone by thesubstance.

By the term “subject in need thereof” is in the present context meant asubject, which can be any animal, including a human being, who isinfected with, or is likely to be infected with, tissue-adheringpilus-forming bacteria which are believed to be pathogenic.

By the term “an effective amount” is meant an amount of the substance inquestion which will in a majority of patients have either the effectthat the disease caused by the pathogenic bacteria is cured or, if thesubstance has been given prophylactically, the effect that the diseaseis prevented from manifesting itself. The term “an effective amount”also implies that the substance is given in an amount which only causesmild or no adverse effects in the subject to whom it has beenadministered, or that the adverse effects may be tolerated from amedical and pharmaceutical point of view in the light of the severity ofthe disease for which the substance has been given.

The route of administration of the substance could be any conventionalroute of administration, i.e. oral, intraveneous, intramuscular,intradermal, subcutaneous etc. The oral route is preferred.

The dosage of such a substance is expected to be the dosage which isnormally employed when administering antibacterial drugs to patients oranimals, i.e. 1 μg-1000 μg per kilogram of body weight per day. Thedosage will depend partly on the route of administration of thesubstance. If the oral route is employed, the absorption of thesubstance will be an important factor. A low absorption will have theeffect that in the gastro-intestinal tract higher concentrations, andthus higher dosages, will be necessary. Also, the dosage of such asubstance when treating infections of the central nervous system (CNS)will be dependent on the permeability of the blood-brain barrier for thesubstance. As is well-known in the treatment of bacterial meningitiswith penicillin, very high dosages are necessary in order to obtaineffective concentrations in the CNS.

It will be understood that the appropriate dosage of the substanceshould suitably be assessed by performing animal model tests, whereinthe effective dose level (e.g. ED₅₀) and the toxic dose level (e.g.TD₅₀) as well as the lethal dose level (e.g. LD₅₀ or LD₁₀) areestablished in suitable and acceptable animal models. Further, if asubstance has proven efficient in such animal tests, controlled clinicaltrials should be performed. Needless to state that such clinical trialsshould be performed according to the standards of Good ClinicalPractice.

Although the preferred way of preventing, inhibiting or enhancing thebinding between pilus subunits and chaperones is to administer asubstance with the above mentioned effects on the chaperone, other waysare possible. For instance, substances interacting with one type ofpilus subunit could also have the effects described above, and for thesame reasons. However, as the interaction with the chaperone is likelyto exert effects on the assembly into the pilus of most, if not all,pilus subunits constituting the intact pilus, it is expected that theinteraction with the chaperone will be the most efficient in terms ofhampering bacterial infectivity.

As will appear from the examples below, most of the data on bindingbetween chaperones and pilus subunits have been obtained by studying theinteraction between the PapD chaperone from E. coli. However, since manytissue adhering bacteria have been found to express pili which sharesubstantial homologies in their C-terminal part, and since substantialhomologies have been demonstrated between the various periplasmicchaperones which until now have been isolated (see table A), it isjustified to assume that some substances and classes of substances willbe capable of interacting with the majority of existing periplasmicchaperones and thus be useful in the treatment and/or prophylaxis ofdiseases caused by the bacteria harbouring these chaperones when thesubstance is administered to patients infected with the bacteria.

Thus, the method of the invention for the treatment and/or prophylaxisis especially intended to be used in patients which are infected bybacteria selected from the group consisting of Haemophilus spp,Helicobacter spp, Pseudomonas aeruginosa, Mycoplasma spp, and allmembers of the Enterobacteriacieae family, including Escherichia spp,Salmonella spp, Bordetella spp, Yersinia spp, Proteus spp and Klebsiellaspp. In this connection, especially the bacteria selected from the groupconsisting of E. coli, Y. pestis, Y. enterocolitica, B. pertussis, K.pneumoniae, S. typhimurium, S. typhi, S. paratyphii, Helicobacterpylori, Proteus mirabilis and Haemophilus influenzae are regarded asinfectants which cause infections which can be treated and/prevented bythe use of the method according to the invention.

Accordingly, in important aspects of the invention, the binding of apilus subunit to a chaperone selected from the group consisting of PapD,FimC, SfaE, FaeE, FanE, Cs3-1, F17D, ClpE, EcpD, Mrkb, FimB, SefB, HifB,MyfB, PsaB, PefD, YehC, MrpD, CssC, NfaE, AggD, and Caf1M is prevented,inhibited or enhanced. It is especially preferred that the binding ofPapD to at least one pilus subunit is affected.

As stated above, in a preferred embodiment of the invention, theprevention, inhibition or enhancement of the binding is accomplished byinteracting with, in the molecular chaperone, a binding site which isnormally involved in binding to pilus subunits during transport of thesepilus subunits through the periplasmic space and/or during the processof pilus assembly.

As mentioned, in connection with the present invention, the bindingmotif between PapD and a peptide which constitute the 19 amino acids ofthe C-terminal of PapG (G1′-19′), a pilus subunit, has been determined.As is described in detail herein, other chaperones share substantialhomologies with PapD in this binding site. Thus, such a binding site isof great interest as a target for drugs which are intended to interactwith periplasmic chaperones. Therefore, in a preferred embodiment of theabove described methods of the invention, the binding site which isaffected is one which binds G1′-19′.

Thus, an important aspect of the invention is a method as describedabove, wherein the binding site is a binding site to which the carboxylterminal part of a pilus subunit is capable of binding, and whichcomprises site points substantially identical to the invariant residuesArg-8 and Lys-112 in PapD, and a polypeptide fragment which is capableof interacting with a β-strand of the carboxyl terminal part of thepilus subunit thereby stabilizing the binding of said subunit at theArg-8 and Lys-112 site points of the binding site. An especiallypreferred aspect of the invention is a method as described above, wherethe binding side is the G-protein binding site of PapD as describedherein.

The term “site-point” refers to a chemical group with well definedphysical/chemical characteristics such as size, charge,hydrophobicity/hydrophilicity, polarity, direction of hydrogen bonds aswell as a 3-dimensional position (distance and angle) relative to othersuch chemical groups. Thus site-points which are “substantiallyidentical” to Arg-8 and Lys-112, are chemical groups which substantiallyshare the same well-defined physical/chemical characteristics as thesetwo amino acids.

The term “invariant residues” refer to amino acid residues which can befound in a number of proteins without there being any variation withregard to the precise type of the amino acid and without there being anysubstantial variation in their function in the proteins. The presence ofinvariant residues in a large number of related proteins normally is anindication of the biological importance of such residues, sincemutations lacking these residues apparently lacks the function of theintact protein, too. As described herein, it has been found that allperiplasmic molecular chaperones share the amino acid residues which areequivalent to Arg-8 and Lys-112. It is believed that these two residuestherefore are of considerable importance to pilus-forming bacteria.

“A polypeptide fragment capable of interacting with a β-strand of thecarboxyl terminal part of a pilus subunit” indicates that part of thechaperone (which is also a part of the binding site) is capable ofinteracting with a β-strand of the pilus subunit. This interactionserves as a stabilizing factor in the binding between the pilus subunitand the chaperone and is considered a very important part of the totalmotif of binding between the chaperone and the pilus subunit. Further,it has recently been rendered probable by the inventors that theβ-strand serves as a template for the correct folding of the pilussubunit (cf. example 10).

As explained herein, the C-terminal part of many, if not all, knownpilus subunits, share substantial homologies, which is anotherindication of the importance of the 3-dimensional structure of the pilussubunit as well as of the chaperone in order for the binding to takeplace and be stable.

As appears from the examples, another binding site residing in domain 2of PapD has been identified. This binding site interacts with fusionprotein MBP-G1′-140′ as well as with a short peptide constituted of theC-terminal amino acid residues 125′to 140′ of PapG. Thus, also thisbinding site is of great interest as a target for drugs which areintended to interact with periplasmic chaperones. Therefore, a preferredembodiment of the above described methods of the invention is a methodwherein the binding site which is affected is one which binds either ofthe two above-described peptides.

It will be understood that the above-described methods comprisingadministration of substances in treating and/or preventing diseases aredependent on the identification or de novo design of substances whichare capable of exerting effects which will lead to prevention,inhibition or enhancement of the interaction between pilus subunits andperiplasmic molecular chaperones. It is further important that thesesubstances will have a high chance of being therapeutically active.

Thus, an aspect of the invention relates to a method for identifying apotentially therapeutically useful substance capable of interacting witha periplasmic molecular chaperone, thereby preventing, inhibiting orenhancing the interaction between a periplasmic molecular chaperone anda pilus subunit, the method comprising at least one of the followingsteps:

1) testing a candidate substance in an assay in which the possibleprevention, inhibition or enhancement by the substance of theinteraction between the periplasmic molecular chaperone and the pilussubunit is determined by

a) adding the substance to a system comprising the periplasmic molecularchaperone or an analogue thereof in an immobilized form and the pilussubunit or an equivalent thereof in a solubilized form and determiningthe change in binding between the pilus subunit or equivalent thereofand the periplasmic molecular chaperone or analogue thereof caused bythe addition of the substance, or

b) adding the substance to a system comprising the pilus subunit or anequivalent thereof in an immobilized form and the periplasmic molecularchaperone or an analogue thereof in a solubilized form and determiningthe change in binding between the pilus subunit or equivalent thereofand the periplasmic molecular chaperone or analogue thereof caused bythe addition of the substance, or

c) adding the substance to a system comprising the pilus subunit or anequivalent thereof as well as the periplasmic molecular chaperone or ananalogue thereof in solubilized form and determining the change inbinding between the pilus subunit or equivalent thereof and theperiplasmic molecular chaperone or analogue thereof caused by theaddition of the substance, or

d) adding the substance to a system comprising the pilus subunit or anequivalent thereof as well as the periplasmic molecular chaperone or ananalogue thereof in solubilized form and measuring the change in bindingenergy caused by the addition of the substance, and identifying thesubstance as potentially therapeutically useful if a significant changein the binding energy between the pilus subunit or equivalent thereofand the periplasmic molecular chaperone or analogue thereof is observed,

and identifying the substance as potentially therapeutically useful if asignificant change in the binding or binding energy between the pilussubunit or equivalent thereof and the periplasmic molecular chaperone oranalogue thereof is observed;

2) testing a candidate substance in an assay in which the possibleprevention, inhibition or enhancement of the interaction between theperiplasmic molecular chaperone and the pilus subunit is determined by

adding the substance to a system comprising living tissue-adheringpilus-forming bacteria followed by determination of the growth rate ofthe bacteria; a reduction in growth rate compared to a correspondingsystem wherein the substance has not been added being indicative ofprevention, inhibition or enhancement of the binding between theperiplasmic molecular chaperone and the pilus subunit, or

adding the substance to a system comprising living tissue-adheringpilus-forming bacteria followed by a determination of the tissueadhesion of the bacteria, a reduction in tissue adhesion compared to acorresponding system wherein the substance has not been added beingindicative of prevention, inhibition or enhancement of the bindingbetween the periplasmic molecular chaperone and the pilus subunit,

and identifying the substance as potentially therapeutically useful if areduction in growth rate or tissue adhesion is observed after theaddition of the substance; and

3) administering, to an experimental animal, a substance which has beenestablished in vitro to prevent, inhibit or enhance the interactionbetween a periplasmic molecular chaperone and a pilus subunit, theexperimental animal being inoculated with tissue-adhering pilus-formingbacteria before, simultaneously with or after the administration of thesubstance, and electing as a substance suitably capable of interactingwith a periplasmic molecular chaperone, a substance preventing and/orcuring and/or alleviate disease caused by the bacteria.

By the term “an equivalent of a pilus subunit” is meant a compound whichhas been established to bind to the chaperone in a manner which iscomparable to the way the pilus subunit binds to the chaperone, e.g. bythe demonstration of the pilus subunit and the equivalent competing forthe binding to the chaperone. Preferred equivalents of pilus subunitsare G1′-19′WT, MBP-G1′-140′ and G125′-140′, which are all described indetail herein.

The term “an analogue of a chaperone” denotes any substance which havethe ability of binding at least one pilus subunit in a manner whichcorresponds to the binding of said chaperone to a pilus subunit. Such ananalogue of the chaperone can be a truncated form of the intactchaperone (e.g. one of the two domains of PapD) or it can be a modifiedform of the chaperone which may e.g. be coupled to a probe, marker oranother moiety. Finally, the analogue of the chaperone can be anisolated, but partially or fully functional, binding site of thechaperone or a synthetic substance which mimics such a binding site.

The immobilization mentioned above may be simple non-covalent binding toan adhering surface or a host or receptor molecule such as an antibody,or covalent binding to a spacer molecule such as a polymer or a peptide.

In the above mentioned step 1a), the pilus subunit or the equivalentthereof being bound to the periplasmic molecular chaperone or ananalogue thereof can be detected in a number of ways, e.g. by the pilussubunit or the equivalent thereof being labelled, or by means of alabelled ligand (such as an antibody) capable of reacting with the pilussubunit or the equivalent thereof, or by means of a refractive indexbased determination of the extend of binding, such as the PharmaciaBiaCore® assay.

Accordingly, in step 1b) the periplasmic molecular chaperone or theanalogue thereof being bound to the pilus subunit or the equivalentthereof may be detected by the periplasmic molecular chaperone or theanalogue thereof being labelled, by means of a labelled ligand (e.g.antibody) capable of reacting with the periplasmic molecular chaperoneor the analogue thereof, or by means of a refractive index baseddetermination of the extend of binding, such as the Pharmacia BiaCore®assay.

In step 1c) the periplasmic molecular chaperone or the analogue thereofbeing bound to the pilus subunit or the equivalent thereof may bedetected by separation of pilus subunit/chaperon complexes (e.g. byultracentrifugation, ultrafiltration, liquid chromatography, such assize exclusion chromatography, or electrophoresis). Described below is amethod relying on the changes in fluorescence of a short PapG fragmentwhen this fragment is bound to PapD. This method is a preferred assay inthe method of the invention.

The determination of binding energy in step 1c) is preferably performedin a microcalorimetric system using the well-known technique ofmicrocalorimetry.

The above-indicated steps serve 3 purposes. The types of assays instep 1) are intended to shed light over the ability of the candidatesubstance of interacting with the chaperone. In the instances whereinlabelled substances, chaperones or antibodies are used, the label couldbe a radioactive label, a fluorescent or light absorbing label, anenzyme such as horse-radish peroxidase, a ligand such as biotin, or anyother conventional labelling system known to the person skilled in theart. The detection of the labelled compound is then dependent on thechoice of label: radioactivity may be measured in a liquid-scintillationcounter, a gamma counter, or any other convenient detection system forradioactivity, enzyme-labels are detected by the presence or absence ofa specific substrate for the enzyme (optical density assessment,chemical reactivity of the remaining substrate or of the product etc.),fluorescent labels may be detected by fluorescence microscopy or simplemeasurement of the fluorescent emission, light-absorbing labels may bedetected by measurement of absorbtion of light of a characteristicwavelength, and biotin may be detected by its binding to streptavidin.

The separation of high molecular complexes by ultracentrifugation orultrafiltration in 1) may be detected by one of the components of thecomplex being labelled as described above; it is thus possible to detectthe ratio between bound and unbound pilus subunit/equivalent, but thedetection step may also rely on the binding of antibodies to one of thecomponents of the complex, and the subsequent detection of thisantibody. Any conventional chromatographic technique may be employed(HPLC, FPLC, size exclusion, etc) The separation by electrophoresis maye.g. be performed by capillary electrophoresis.

The assays in step 2) all relate to the effects of the candidatesubstance on bacterial activity in vitro. The demonstration of areduction in growth rate of the bacteria or a demonstration of reducedadherence to cells or synthetic surfaces in an assay of course cannot becontributed to the effect of interaction with chaperones only, but ademonstration of this kind should provide a good estimate of thepotential therapeutical usefulness of such a substance.

The determination of growth rate may be performed by counting ofcolonies on solid agar plates striped with the bacteria, by countingbacterial density in liquid growth media (OD₆₀₀ determination), bymeasuring fluorescence of substances such as NAD(P)H, ATP, or aminoacids, which are contained in the bacterial cells only, or by any otherconvenient detection system known to the person skilled in the art. Thedetermination of adherence of the bacteria may be performed in a similarmanner after the adhering bacteria have been isolated. A determinationof adherence is preferably performed by measuring the ability of thebacteria to agglutinate red blood cells or receptor-coated latex beads,by measuring the bacterial adhesion to receptor-coated microtiterplates, or by measuring the bacterial adhesion to other syntheticsurfaces.

In a preferred embodiment of the method described above, the living,tissue adhering pilus-forming bacteria are of a protease deficientstrain, the protease being one which is at least partially responsiblefor the degradation of pilus subunits. One especially preferred type ofstrain is the degP41 strain of E. coli. As described herein, the degP41strain lacks activity of the DegP protease which is responsible fordegradation of pilus subunits in the E. coli when these are not rescuedinto periplasmic space by PapD, and degP41 strains are thus especiallysensitive to changes in the efficiency of PapD, as the accumulation ofpilus subunits is toxic to the cell. It is believed that equivalentproteases exist in other pilus expressing bacteria.

The animal study in step 3) is performed in order to demonstrate thepotential therapeutic usefulness of the candidate substance in vivo.Further, as already mentioned above, such animal studies should alsoestablish the a priori values regarding effective dosage and toxicitybefore the candidate substance finally is tested in human beings incontrolled clinical trials. The animal studies should also provideinformation regarding the convenient formulation of the substance in apharmaceutical preparation as well as the preferred route ofadministration, as it is possible to obtain, from the animal model, datafor absorbtion of the substance as well as data for the metabolism andexcretion of the substance. The experimental animal is preferably amouse, a rat, a cat, a dog, a monkey, a horse, a cow, a pig, or achicken.

The term “suitably capable of interacting with a molecular chaperone” isintended to indicate that a substance, apart from being capable ofinteracting with a molecular chaperone, also is capable of exertingeffects in an in vivo system, i.e. that the substance in addition to itsbinding capability also exhibits compatibility with a biological system,i.a. a patient.

Although the above-indicated in vivo studies, especially the experimentsin animal models, are the best indicators of the potential therapeuticalusefulness of a substance in the prevention, inhibition, or enhancementof the binding between a chaperone and a pilus subunit, it should not beforgotten that the in vitro assays outlined above serve as importantleads when developing compounds with a therapeutical potential. If onerelied only on in vivo assays, it is very likely that compounds which infact exhibit the desired effect on the chaperone/pilus subunitinteraction would be screened out by the in vivo assays, because thesecompounds could lack e.g. the ability to penetrate biological membranes.When using the in vitro assays, a much greater chance of finding a leadcompound is maintained.

The evaluation of the effect of a substance tested in the in vitroassays described herein (cf. in this connection especially the examples)depends on a number of factors. It will be understood by the skilledperson that a small molecule could be added in rather high molarconcentrations in order to exert an effect on the chaperone/pilussubunit interaction (and even then the small molecule may still be aninteresting lead compound), whereas larger molecules may exert markedeffects even in rather low molar concentrations. In general, when any invitro assay described herein is regarded as having a positive resultwhen testing a candidate substance (i.e. that the substance tested showsa “significant” effect), the following condition should be fulfilled:The compound should exert a significant effect on pilussubunit/chaperone interaction (or on an interaction in an equivalentsystem which correlates well to pilus subunit/chaperone interaction),the significant effect being one which with no doubt can be attributedto the interaction between the substance and the chaperone and which isnot an unspecific interaction between the chaperone and the substance(due to e.g. radical changes in the physical and chemical environmentwhen the substance is added). One way of excluding unspecificinteractions as the reason for the exerted effect is to use at least onecontrol which is a chemically comparable substance (with respect tomolecular mass, charge/polarity and gross 3-dimensional conformation(globular, fibrillar etc.). If the control does not result insubstantially the same effect in the assay as the substance, it can beconcluded that the substance must be regarded as an assay positivesubstance.

The assays described in the examples are all good examples of assaytypes, which could serve as the test system in the above-describedmethod of the invention. However, it is preferred that the methoddescribed in example 10 employing a fluorescence labelled variant of apilus subunit is used in step 1c). This assay may shortly be described as follows:

adding the substance to a first system comprising the periplasmicmolecular chaperone or an analogue thereof,

subsequently adding a pilus subunit or an equivalent thereof which hasbeen labelled with an environmentally sensitive fluorescent probe,

determining the fluorescent emission at a particular wavelength which isindicative of the amount of binding between the periplasmic molecularchaperone or the analogue thereof and the pilus subunit or theequivalent thereof, and

comparing the determined fluorescent emission to fluorescent emissiondetermined in a corresponding second system containing substantially thesame concentrations of the molecular chaperone or the analogue thereofand the pilus subunit or the equivalent thereof but substantially nosubstance,

a significant difference in fluorescent emission between the first andsecond system being indicative of interaction between the periplasmicmolecular chaperone or the analogue thereof and the substance.

The advantage of this assay is that it may be employed for quantitativedeterminations of the effect of the tested substance on thechaperone/pilus subunit system. By using this assay the inventors havee.g. determined the constant of binding between a PapG analogue andPapD. The quantitative determinations may be performed by performing thedetermination of fluorescent emission in the second system a pluralityof times at varying molar ratios between the pilus subunit or theequivalent thereof and the periplasmic chaperone and the equivalentthereof, whereupon the constant of binding between the pilus subunit orequivalent thereof and the periplasmic molecular chaperone or analoguethereof is assessed from the determined fluorescent emission data. Fromthe data obtained in this way it is also possible to determine thebinding constant of the substance in a parallel manner, which willappear from claim 11.

It will be understood that the above-indicated method for identifying apotentially therapeutically useful substance is dependent on the actualpresence of the substance. Normally, it is necessary to either purify orsynthesize the candidate substance before it is subjected to theabove-mentioned method. However, since many such candidate substancesare likely to be tested before a substance which is suitably capable ofinteracting with a chaperone will be identified, it is of interest toidentify such substances before they are subjected to the method above,thereby diminishing the resources spent on purification and/or synthesissteps.

Hence, the invention also relates to a method for identifying and/ordesigning a substance, X, capable of interacting with a chaperone, e.g.binding to the chaperone, with a predicted binding energy equal to orbetter than a predetermined threshold value, the method comprising

1) selecting a substance, A, which could potentially interact with asite in the chaperone, and providing a 3-dimensional structuralrepresentation thereof,

2) predicting the binding free energy between the substance A and thesite in the chaperone,

3) if the predicted binding free energy between the substance A and thesite in the chaperone is equal to or better than the predeterminedthreshold value, then identifying the substance A as the substance X,

4) if the predicted binding free energy between the substance A and thesite in the chaperone is not equal to or better than a predeterminedthreshold value, then modifying the 3-dimensional structuralrepresentation and predicting the binding free energy between the thusmodified substance, B, and the site in the chaperone, and

5) repeating step 4 until the predicted binding free energy determinedbetween the resulting substance, X, and the site in the chaperone isequal to or better than the predetermined threshold value.

It is possible to expand the above-mentioned method with two furthersteps, wherein the actual binding free energy is determined, in order toestablish that the experimental binding free energy also is better thanthe predetermined threshold value. By performing the following two steps

6) providing a sample of the chemical substance X and a sample of thechaperone and measuring the binding free energy between the chemicalsubstance X and the chaperone (e.g. by microcalometry as mentionedabove), and establishing that the measured binding free energy betweenthe chemical substance X and the chaperone is equal to or better thanthe predetermined threshold value, and optionally

7) subjecting the substance X to the method mentioned above foridentifying a substance suitably capable of interacting with achaperone, in order to verify that the substance X is a potentiallytherapeutically useful substance capable of interacting with achaperone, it is thus verified that the binding free energy between thecandidate substance and the chaperone actually is better than thepredetermined threshold value. Step 7) further establishes that thecandidate substance stands good chances of being therapeutically useful.

The phrase “predicting the binding free energy” is meant to imply thatthe binding free energy is determined by calculation rather than byperforming experimental work determining the actual binding free energy.One (theoretical) way of predicting binding free energy is by performingfree energy perturbation (FEP) calculations on the interactingsubstances, but because of the vast amount of calculations such anapproach would have as a result it is preferred that the empiricalapproximative method described below is employed.

The term “better than” is intended to mean that the binding free energyhas a value which is higher than the binding free energy which has beenchosen as the threshold value, meaning that the ΔG is numerically higherthan the threshold value selected. Or in other words: The term isintended to mean that the binding between the substance and thechaperone is more favourable energetically than the situation were thesubstance and the chaperone are suspended independently in solution.

In order to predict the binding energy in the above-indicated method,according to the invention it is especially preferred to use thefollowing method:

Assessing the average energy difference, <ΔV_(X−s) ^(el)>, defined as<V_(X−s) ^(el)>_(B)−<V_(X−s) ^(el)>_(A), between the contribution frompolar interactions to the potential energy between the chemicalsubstance X and its surroundings (denoted s) in two states, one state(A) being where the chemical substance is surrounded by solvent, theother state (B) being where the chemical substance, bound to aperiplasmic molecular chaperone or an analogue thereof, is surrounded bysolvent, assessing the average energy difference, <ΔV_(X−s) ^(vdw)>,defined as <V_(X−s) ^(vdw)<_(B)−<V_(X−s) ^(vdw)>_(A), between thecontribution from non-polar interactions to the potential energy betweenthe chemical substance X and its surroundings (denoted s) in two states,one state (A) being where the chemical substance is surrounded bysolvent, the other state (B) being where the chemical substance, boundto a periplasmic molecular chaperone or an analogue thereof, issurrounded by solvent, and

calculating the absolute binding free energy as an adjusted combinationof the two above-mentioned average energy differences.

In the mathematical equations herein, the symbol <>means moleculardynamics average. The index X-s means compound-solvent (orcompound-surrounding), the letter “X” denoting the chemical substance X.Normally the substance X will function as an inhibitor of the bindingbetween the periplasmic chaperone and pilus subunits, but as discussedherein, it is also a possibility that the compound or drug will affectthe chaperone in such a way that the binding between pilus subunits andthe chaperone is enhanced. The superscript “el” designates the polar orelectrostatic energy, while the superscript “vdw” indicates “van derWaals”, another designation for the non-polar interactions. The symbol Δindicates that the quantity in state A is subtracted from the quantityin state B.

In the present context the term “an analogue of a periplasmic molecularchaperone,” should be understood, in a broad sense, any substance whichmimics (with respect to binding characteristics) an interesting-part ofa periplasmic molecular chaperone (e.g. the pilus subunit bindingpart(s)), and the interaction of which with a chemical substance or agroup or plurality of chemical substances, e.g. drug candidates, is tobe studied. Thus, the analogue may simply be any other chemical compoundregarded as capable of interacting with the chemical substance in amanner which mimics the binding between the chaperone and a pilussubunit in vivo, but most often the analogue will be a relatively largemolecule, in other words a macromolecule such as a protein or anoligonucleotide, which is relatively large compared to the chemicalsubstance; although the chemical substance interacting with theanalogue, of course, in itself be a macromolecule. In the presentcontext, the periplasmic molecular chaperone or analogue thereof ispreferably the periplasmic chaperone or an analogue thereof whichexhibits at least one interesting binding characteristic relevant forthe assembly of pili.

The basis for the above-indicated approach for determining the bindingfree energy is explained in the following:

As a starting point is taken the linear response approximation forelectrostatic forces which for polar solutions as a result yieldsquadratic free energy functions in response to the development ofcharges. This is, e.g., the familiar result from Marcus' theory ofelectron transfer reactions (Marcus, 1964). For a system with twostates, A and B, given by two potential energy functions V_(A) and V_(B)one obtains, within the approximation of harmonic free energy functionsof equal curvature, the relationship (see Lee et al., 1992 andreferences therein):

λ=<V _(B) −V _(A)>_(A) −ΔG _(AB) =<V _(A) −V _(B)>_(B) +ΔG _(AB)  (a)

where ΔG_(AB) is the free energy difference between B and A, λ thecorresponding reorganisation energy and <>_(i) denotes an averageevaluated near the minimum of the potential i. Thus,

ΔG _(AB)≅½(<ΔV> _(A) +<ΔV> _(B))  (b)

where ΔV now denotes the energy difference V_(B)−V_(A). If the hydrationof a single ion is considered, this can be shown to give ΔG_(sol)^(el)=½<V_(X−s) ^(el)>, i.e. that the electrostatic contribution to thesolvation energy equals half of the corresponding ion-solventinteraction energy (Warshel and Russell, 1984; Roux et al., 1990).Returning now to the binding problem, this result may be exploited inthe following manner: For each salvation process, i.e. salvation of thesubstance in water and inside the protein, two states are consideredwhere the first has the substance in vacuum and a non-polar cavity(given, e.g., by Lennard-Jones potential) already made in the givenenvironment. The second state corresponds to the intact substancesurrounded by water or the solvated protein. The linear responseapproximation will then again give that ΔG_(bind) ^(el)≅½<V_(X−s)^(el)>, where V_(X−s) ^(el) is the solute-solvent electrostatic term.Hence, the electrostatic contribution to the binding free energy can beapproximated by ΔG_(bind) ^(el)≅½<V_(X−s) ^(el)> (where the Δ now refersto the difference between protein and water) and thus obtained from twoMD simulations of the solvated substance and of the substance-proteincomplex.

The validity of the linear response results in the case of ionicsolvation has been confirmed, e.g., in the study by Roux et al. (1990).Some additional calculations were also performed on simple systems thatcorroborate the approximation of equation b. These tests were carriedout by comparing the free energy obtained from FEP/MD simulations ofcharging Na⁺ and Ca²⁺ ions in a spherical water system ({dot over(A)}qvist, 1990) with the corresponding <V_(X−s) ^(el)> from 75 ps MDtrajectories. This yielded factors relating <V_(X−s) ^(el)> to ΔG_(sol)^(el) of 0.49 for Na⁺ and 0.52 for Ca²⁺, both values being close to thepredicted result of ½. A similar test on the charging of a methanolmolecule, given by the OPLS potential (Jorgensen, 1986) in water gave aΔG_(sol) ^(el)/<V_(X−s) ^(el)> ratio of 0.43.

A crucial question is how to account for the contribution of non-polarinteractions and hydrophobic effects to the free energy of binding whichwas termed ΔG_(bing) ^(vdw). In the ideal case, it should be possible toestimate this contribution from the non-polar (or van der Waals)interaction energies. The liquid theories of Chandler and coworkers(Chandler et al., 1983; Pratt and Chandler, 1977) have been successfullyused to analyze hydrophobic effects and to calculate free energies oftransfer for some non-polar molecules (Pratt and Chandler, 1977), but noanalytical treatment of that kind seems possible for salvation in aninhomogeneous environment such as a protein's active site. However, ithas been noted that the experimental free energy of salvation forvarious hydrocarbon compounds, such as n-alkanes, depends approximatelylinearly on the length of the carbon chain both in their own liquids aswell as in water (Ben-Naim and Marcus, 1984). MD simulations ofn-alkanes solvated in water and in a non-polar van der Waals solventhave been carried out, which indicate that also the averagesolute-solvent interaction energies vary approximately linearly with thenumber of carbons in the chain (the relationships being different indifferent solvents, of course). It thus seem possible that a simplelinear approximation of ΔG_(bind) ^(vdw) from <ΔV_(X−s) ^(vdw)> might beable to account for the non-polar binding contribution. For instance, ifσ is considered some appropriate measure of the size of the solute andif the solute-solvent van der Waals interaction energies and thecorresponding non-polar free energy contributions (both in water andprotein) depend linearly on σ, such that

<V _(p) ^(vdw)>=α_(p) σ, <V _(w) ^(vdw)>=α_(w) σ, ΔG _(p) ^(vdw)=β_(p)σ

and

ΔG _(w) ^(vdw)=β_(w)σ

then${\Delta \quad G_{bind}^{vdw}} = {\frac{\beta_{p} - \beta_{w}}{\alpha_{p} - \alpha_{w}} < {\Delta \quad V_{X - s}^{vdw}} >}$

is obtained. Since it seems difficult to derive a factor relating thetwo quantities in a reliable way from purely theoretical considerations,the approach is taken to empirically try to determine such arelationship which is capable of reproducing experimental binding data.Thus, the free energy of binding is in one embodiment of the inventionapproximated by

ΔG _(bind)=½<ΔV _(X−s) ^(el) >+α<ΔV _(X−s) ^(vdw)>  (1)

the parameter α being determined by empirical calibration.

Although, as discussed above, a theoretical prediction of thecoefficient for <ΔV_(X−s) ^(el)> is ½, it may be practically useful toalso treat this coefficient as an empirical parameter. This would leadto the free energy of binding being approximated by

ΔG _(bind) =β<ΔV _(X−s) ^(el) >+α<ΔV _(X−s) ^(vdw)>  (1b)

where both parameters, α and β, are determined by empirical calibration.

Finally, in some cases, it seems suitable to add an additional constantterm to Equation 1, so that the equation becomes

ΔG _(bind)=½<ΔV _(X−s) ^(el) >+α<ΔV _(X−s) ^(vdw) >+c  (2)

where c is a constant reflecting extrapolation to zero size of thechemical substance, that is, where the regression line is distinctlyoffset from origin when moving towards zero size of the chemicalsubstance. The parameter c may also be used to correct for possiblesystematic errors due to e.g. the neglect of induced polarisation,possible force field deficiencies etc. In these cases, c will normallyassume a value between −10 and 10 kcal/mol, typically between −3 and 3kcal/mol, such as between −2 and 2 kcal/mol, e.g. between −1 and 1kcal/mol. However, it is anticipated that in many cases, c can suitablybe set to zero, as the extent of deviation will be of minor importancefor the usefulness of the predicted values.

If also the electrostatic coefficient i treated as an empiricalparameter, the approximation of the binding free energy assumes its mostgeneral form, namely

ΔG _(bind) =β<ΔV _(X−s) ^(el) >+α<ΔV _(X−s) ^(vdw) >+c  (2b)

where now both α, β and c are to be determined by empirical calibration.

While the solvent used in the above method is suitably and most often anaqueous solvent like water, it is within the scope of the invention totake any other suitable solvent as a starting point, including, e.g.,methanol, ethanol, acetone, acetonitrile, chloroform, hexane, etc., ormixtures thereof or combinations of such solvents or mixtures thereofwith water. The selection of the solvent will be of little importance tothe predicted values as long as the solvent is one which is able todissolve or solvate the receptor molecule and the substance (in thepresent context this means that a sufficient amount of the periplasmicmolecular chaperone or analogue thereof can be homogeneously mixed withthe solvent without precipitation so as to allow the determination ofbinding energies by some suitable method), but there may be cases whereit is advantageous to modify the solvent environment (e.g. by modulatingthe ionic strength) in which the interaction of the substance and thereceptor molecule is to take place. If the environment in which theinteraction between the chemical substance, such as a drug, and aperiplasmic molecular chaperone or an analogue thereof is to take placein the actual use of the drug is the human body, it might beparticularly suitable to imitate e.g. human plasma as the solvent.

A thorough discussion of the above-referenced method for determining thebinding free energy between two molecules can be found in InternationalPatent Application No. PCT/IB94/00257 and in {dot over (A)}qvist et al,1994. These two documents are hereby incorporated by reference.

The above referenced method for determining the binding free energy hasbeen employed in example 3 in order to identify compounds which stands ahigh chance of binding to the binding site of PapD; this means thatcalculations as the above described have been performed as the lasttheoretical step before compounds have actually been synthesized.

It will be understood that the above mentioned methods for identifyingsubstances capable of interacting with chaperones will prove especiallyefficient in identifying substances which are of potentialpharmaceutical value if the site to which they bind is known to beinvolved in pilus assembly.

Therefore, it is preferred that the site with which the substance maypotentially interact, and to which the binding free energy is predicted,is the pilus subunit binding part of a molecular chaperone, such as thepilus subunit binding site of a molecular chaperone selected from thegroup consisting of PapD, FimC, SfaE, FaeE, FanE, Cs3-27, F17D, ClpE,EcpD, Mrkb, FimB, SefB, HifB, MyfB, PsaB, PefD, YehC, MrpD, CssC, NfaE,AggD and Caf1M, or an analogue of such a pilus subunit binding site,since the pilus subunit binding sites in these chaperones show extensivehomologies. It is especially preferred that the binding site is thepilus subunit binding site of PapD or an analogue thereof.

As will appear from the examples, an important part of the chaperonebinding motif has been discovered and a peptide corresponding to thismotif has been synthesized and co-crystallized with PapD to provide astructural basis for the mechanism of action of PapD. The moleculardetails of the PapD-adhesin recognition interface clearly demonstratethe function of the conserved cleft in the entire pilus chaperonesuperfamily in subunit binding and in shuttling virulence determinantsto the surface of pathogenic bacteria. The PapD-peptide crystalstructure essentially represents a “snapshot” of a fundamental processin bacterial pathogenesis: the interaction of an adhesin with achaperone, which is a prerequisite to adhesin presentation on themicrobial surface.

Thus, the inventors of the present invention have by the use of X-raycrystallography elucidated the mechanism of binding between PapD and thepilus subunit PapG thereby identifying an essential part of a definedbinding site responsible for the binding between pilus subunits andtheir periplasmatic chaperones, and thus providing a method to enabledrug design of chaperone inhibiting anti-bacterial compounds.

Having determined the location of a promising binding site forinhibitory ligands as described above (see details in examples 1 and 2),the computer programs “PLIM” and “PLIM_DBS” (developed by Symbicom AB)have been used to find templates for families of compounds capable ofbinding to the binding site.

PLIM is a Protein Ligand Interaction Modeller that constructs putativeligands for a protein using thermodynamic criteria. It calculates theenergy of interaction between the protein and sample probes that aresuccessively placed at different points on a regular grid around themolecule. For each position and orientation the interaction energybetween the probe and the atoms of the protein is calculated. Theenergies are stored, and the best positions for a particular probe arewritten out (the basic calculations are described by Goodford (1985) andBoobbyer (1989) and implemented in the commercially available programGRID; the PLIM implementation is somewhat different in that the energyvalues are converted to discrete points that are associated with thechemical probe, enabling easy output to e.g. data base searchingprograms). The program then builds up the ligand by incorporatingselected probe atoms at positions of energy minima on the grid. The userselects which atoms and groups should be used as probes, and whichcriteria should be used to determine those that will be incorporatedinto the ligand. The energy is calculated as the sum of electrostatic,Van der Waals and hydrogen-bonding contributions as described herein.

The PLIM runs result in a number of suggested positions and orientationsof favourable chemical groups in the region near the binding site. Thesegroups which have physical properties like charge, hydrogen bondingdirectionality and extended atom radia, will hereafter be denoted “sitepoints”.

A search for potential ligands is then made by searching a database forknown molecular structures that match the positions of these groups ofsite points, using PLIM_DBS.

The core of PLIM_DBS is an algorithm for subgraph isomorphism (cf.Ullman (1976) and Brint (1987)), where three sitepoints are representedas a distance matrix (“the pattern matrix”). The program looks for thisdistance pattern in the distance matrix formed from every entry in thedatabase. If the pattern is found, the entry is superimposed on to thesitepoints and if the corresponding atom types match the entry and itsorientation is saved in a hit-list. Added to this basic scheme a numberof options regarding surface complementarity can be used, i.e. onlyentries which are matching the protein surface with respect tohydrophobic and steric properties are saved.

PLIM_DBS is thus a database searcher which hunts through a collection of3-dimensional molecule coordinate sets, looking for entries that containa certain pattern of atoms. This pattern is specified in terms of atomtype, and of spatial position and orientation; for instance a search maybe made for compounds containing an sp3 carbon atom that is 4.2 Å from asp2 oxygen and 5.1 Å from a hydroxyl group that in turn is 5.6 Å fromthe oxygen. The strictness of the search can be adjusted by the user byvarying the tolerance on the distance criteria and the atom-typematching, determining, for instance, whether a sp2 carbon that is alittle more than 4.2 Å from an oxygen should be considered as a hit.Those hits that are found are then ranked according to a score thatreflects how well the target atoms superimpose on the real molecule, andalso on how complementary the molecular surface of the compound is tothat of the binding pocket of the protein.

The result from a PLIM_DBS search is a list of molecular structures andtheir atomic coordinates, superpositioned on to the sitepoints, andgiven a score (“goodness of fit”).

The procedure does not try to optimize the positioning of thestructures, nor does it perform any molecular mechanics or dynamicscalculations. Both protein and the extracted structures are treated asrigid bodies.

The structures from the database search are displayed in the context ofthe protein and its surface on a graphics system using a commonlyavailable molecular modelling package. Usually the structures show someunfavourable interactions with the protein, or lack groups to fill oute.g. hydrophobic pockets. Hence, the structures form the database searchare regarded as templates, to be modified and improved by an organicchemist. This process also involves choosing compounds which are easy tosynthesize, which is of particular interest if the synthesis capacity islimited.

The best of these database hits are thus examined visually using acomputer-graphic modelling system, and the most promising of these areselected according to a wealth of physico-chemical reasoning.

The templates are modified using a small molecule 3D builder (MacMimic).Each template gives rise to a compound class, e.g. denoted “hdo”. Eachmodification assigned a specific number (e.g. hdo_(—)3) and thecoordinates and a description are stored in a tree structure, using theprogram ARVS_JAKT developed by Symbicom. The design is performed in acollaboration between protein structure experts and organic chemists, inorder to provide the best tools possible for the chemists who willactually synthesize the compounds.

The efficacy of these modifications is finally assessed usingmolecular-dynamics free energy calculations as described herein to studythe stability of the protein-ligand complex ({dot over (A)}qvist et al.,1994; {dot over (A)}qvist and Medina, 1993).

In order to maximize the efficiency of the above-mentioned methods foridentifying/designing substances which are capable of interacting with amolecular chaperone, it is preferred that the substance A is likely tobe a substance which is capable of binding to the selected binding site.

In view of the above-described modus operandi for selecting substanceswhich should interact with chaperones like PapD, this can, according tothe invention, be accomplished when the substance A is selected byperforming the following steps:

co-crystallizing the periplasmic molecular chaperone or the analoguethereof with a ligand capable of interacting with a site in theperiplasmic molecular chaperone or the analogue thereof and establishingthe three-dimensional conformation of the periplasmic molecularchaperone or the analogue thereof and the ligand when interacting bymeans of X-ray crystallography,

using the above-established conformation of the periplasmic molecularchaperone or the analogue thereof to establish a 3-dimensionalrepresentation of the site in the periplasmic molecular chaperone or theanalogue thereof interacting with the ligand during binding,

selecting a number of distinct chemical groups, X1, and determining thepossible spatial distributions of the X1 chemical groups which maximizesthe binding free energy between the chemical groups and the site in thechaperone or the analogue interacting with the ligand,

extracting, from a database comprising three-dimensional representationsof molecules, a molecule which has the X1 chemical groups in thepossible spatial distributions determined above,

optionally modifying the 3-dimensional representation of the moleculeextracted from the database, and identifying the optionally modifiedmolecule as the substance A.

According to the invention the above-indicated steps are especiallypreferred when the ligand is a pilus subunit or a part thereof withwhich the chaperone normally interacts during transport of the pilussubunit through the periplasmic space and/or during pilus assembly.

By the use of the above-mentioned method for identifying substancescapable of interacting with the periplasmic chaperones, several classesof substances have been identified which have proved promising in tiedesign phase.

Drug design efforts for inhibitors/enhancers to PapD have concentratedon the region of the molecule where the G-peptide is observed to bind.This region will now be described in detail, using one of the putativeinhibitors (bpy_(—)9, see below) as a reference structure.

The binding site is dominated by the central charged side chains ofArg-8 and Lys-112, which bind to the sulphate moiety of bpy_(—)9.Adjacent to this is a small, shallow hydrophobic pocket formed by theside-chains of Ile-154, Ile-194, and Thr-7, against which the 2-ethylgroup of bpy_(—)9 packs (Pocket 1). A group as large as a phenyl ringcould be accommodated here, attached to sugar position 2, and withpossible substituents that could receive or donate a hydrogen bond toThr-7, or donate to the backbone carbonyl of 198.

There is also a larger pocket comprising residues Leu-4, Ile-111, Thr-7and Thr-109, in which the phenyl ring of bpy_(—)9 nestles (Pocket 2). Agroup as large as naphthalene could be substituted on the 6-position ofthe carbohydrate scaffold to fill this sub-site, with substituents thatcould form a hydrogen bond to Thr-109, or to any of the polar backboneatoms of residues Leu-4, Arg-6, or Lys-110. Then there is a long,shallow patch that includes Tyr-87, and the aliphatic regions of Lys-110and Lys-112, which could accommodate a tricyclic system such as2-phenanthryl substituted on position 3 of the sugar (Patch 3).Substituents to hydrogen bond to Tyr-87 or the backbone of Lys-110 canbe considered, as well as negatively charged groups to complement theside chains of Lys-110 and Lys-112. Tyr-87 is a potentialcharge-transfer donor to a electron deficient π-system such as anitroaryl.

Models of several homologous chaperones (SfaE, MrkB, HifB and FimC) havebeen made from the PapD structure, and differences between the modelstructures has influenced the design of inhibitors, such that proposedligands should bind to all the structures looked at. For example, it istempting to complement the charged side chain of Arg-200 with an acidicgroup in the ligand, but since two of the other structures have an Aspat this position, the residue is not considered as a good candidate.Arg-8 is fully conserved, as is Lys-112, Thr-7 and Ile-11. Tyr-87becomes a Trp in three structures, but the overall nature of patch 3 isnot changed by this. PapD is actually alone with its Thr-Lys sequence at109-110, all of the four other structures being Ser-Arg here, but again,these conservative changes do not significantly alter design criteria.

Although substances which interact with the binding site responsible forbinding to G-proteins are obvious candidates as inhibitors/enhancers ofperiplasmic chaperones, it will be understood that molecules capable ofinteracting with other sites in periplasmic chaperones are interestingin this aspect, too. It is highly possible that an interaction withanother site than the one binding G-protein in i.e. PapD may cause PapDto either be prevented, inhibited or enhanced in its action as aperiplasmic chaperone.

As mentioned above, a family of substances (called the bpy familyherein) is an important aspect of the invention. Thus, the inventionrelates to novel compounds of the general formula:

wherein

V₁ is O, S, SO, SO₂, CH₂, C(OH)H, CO or CS;

W₁ is O, S, SO₂, SO₃, CH₂ or NH;

R₁ is H; C₁₋₂₄ alkyl, C₁₋₂₄ alkenyl or C₁₋₂₄ alkynyl, which alkyl,alkenyl and alkynyl may be substituted with one or more substituentsindependently selected from OH, —CONH₂, —CSNH₂, —CONHOH, —CSNHOH,—NHCHO, —NHCONH₂, —NHCSNH₂, —NHSO₂NH₂ and —SO₂NH₂; acyl; or—(CH₂CH₂O)_(s)—H, wherein s=1,2,3;

R₂ is a group of the formula

wherein

A is —CH—(CH₂)_(n)—, or —CH═CH—(CH₂)_(n−1)—(n>0) or

B is —(CH₂)_(m)— or ═CH—(CH₂)_(m−1)— (m>0);

X₂ is N, CH or C (when B is ═CH—(CH₂)_(m−1)—; and

Y₂ is O, S, NH, H₂ or H (n=1); and

4>m+n>0, n<3, and m<3;

or R₂ is a group of the formula

wherein

A is —CH—(CH₂)_(n)—, or —CH═CH—(CH₂)_(n−1)— (n>0) or

B is —(CH₂)_(m)— or ═CH—(CH₂)_(m−1)— (m>0); and

X′₂ is O, NH, CH₂ or S (when p=0); N or CH— (p=1);

or C (when p=1 and B is ═CH—(CH₂)_(m−1)—);

V₂, Z₂ and W₂ are independently H, OH, —CONH₂, —CSNH₂, —CONHOH, —CSNHOH,—NHCHO, —NHCONH₂, —NHCSNH₂, —NHSO₂NH₂, —SO₂NH₂, or V₂ and Z₂, or Z₂ andW₂ together form —NHC(O)NH—, —C(O)NHC(O)—, —NHS(O₂)NH—, —C(O)NHO—,—C(S)NHO—, —S(O₂)NHO—, or —S (O₂)NHC (O)—;

4>m+n>0, n<3, and m<3;

or R₂ is a group —W₅—(C₁₋₅ alkyl or C₂₋₅ alkenyl or C₂₋₅ alkynyl)wherein W₅ is a bond or is selected from —O—, —S—, —SO₂—, and —NHC(O)—,and the C₁₋₅ alkyl, C₂₋₅ alkenyl or C₂₋₅ alkynyl moiety may besubstituted with up to three groups selected independently from OH,—CONH₂, —CSNH₂, —CONHOH, —CSNHOH, —NHCHO, —NHCONH₂, —NHCSNH₂, —NHSO₂NH₂and —SO₂NH₂;

—Z₁—R₃ is —SO₂(OH), —PO(OH)₂, —OSO₂(OH), —NHSO₂(OH), —NH—CO—COOH,—SPO(OH)₂, —CH₂COOH, tetrazol-5-yl or tetrazol-5-ylmethyl, or saltsthereof;

or Z₁ is —O—, —S—, —NH—, or —CH₂—, and R₃ is a group of the formula:

D is —CH₂—, —CO—, —SO₂—, —NH—SO₂—, —NH—CO—, —O—PO(OH)— or a saltthereof;

Z₃ is H, OH, —CONH₂, —CSNH₂, —CONHOH, —CSNHOH, —NHCHO, —NHCONH₂,—NHCSNH₂, —NHSO₂NH₂—, —SO₂NH₂, —SO₂(OH), —PO(OH)₂, —OSO₂(OH),—NHSO₂(OH), —COOH, tetrazolyl-5-yl or tetrazolyl-5-ylmethyl or a saltthereof,

with the proviso that when D is —CH₂—, —CO—, —SO₂—, —NHSO₂— or —NHCO—,then Z₃ is —SO₂(OH), —PO(OH)₂, —OSO₂(OH), —NHSO₂(OH), —COOH,tetrazolyl-5-yl or tetrazolyl-5-ylmethyl or a salt thereof;

X₃ and Y₃ independently are H, NO₂, SO₂NH₂, CONH₂, CF₃ or F; and

U₄-W₄ is —CHCH—, —CH₂CH₂—, —C(OH)CH—, —CHC(OH)—, —CH(OH)CH₂—,—CH₂CH(OH)—, —CH(OH)CH(OH)—, —C(O)NH—, —NHC(O)—;

Y₁ is —O— or —S—;

R₄ is H or, when Y₁ is S, S (CH₂)_(q)N(R₉)₃ ⁺ and q is an integer 2-4,where R₉ H or C₃;

R₅ is H; C₁₋₆ alkyl, C₂₋₆ alkenyl or C₂₋₆ alkynyl, and the C₁₋₆ alkyl,C₂₋₆ alkenyl or C₂₋₆ alkynyl moiety may be substituted with OH, —CONH₂,—CSNH₂, —CONHOH, —CSNHOH, —NHCHO, —NHCONH₂, —NHCSNH₂, NHSO₂NH₂ or—SO₂NH₂; or aryl, aryl(C₁₋₂)alkyl, heterocyclyl, orheterocyclyl(C₁₋₂)alkyl which may optionally be substituted in the arylor heterocyclyl moieties with one, two or three substituents selectedindependently from OH, F, Cl, NH₂, CONH₂, NHCOH, and SO₂NH₂;

X₁ is —O—, —S—or —NH—;

R₆ is H or, when X₁ is NH, acyl, HOCNH-Val-Met-,HOCNH-Ile-(S,S)-dioxo-methionyl- orHOCNH-Val-(pyran-4-on-2-yl)-ala-nyl-;

or a salt of such a compound.

Another family of substances called the hdo-family has also beensynthesized. Hence, the invention also relates to novel compounds of thegeneral formula

wherein

X is O, P, P(O), S, SO, SO₂, CH₂, C(OH)H, or a group NQ₁₁, wherein Q₁₁is H, OH, C₁₋₂₄ acyl or C₁₋₂₄ alkyl;

Z₁₁ is a bond, O, CH₂, S, SO, SO₂, or a group NQ₁₂, wherein Q₁₂ is H,C₁₋₂₄ acyl or C₁₋₂₄ alkyl;

R₁₁ is H; C₁₋₂₄ alkyl, C₂₋₂₄ alkenyl, or C₂₋₂₄ alkynyl, which may besubstituted with one or more substituents independently selected from—OH, —COOH, —F, —Cl, —CONH₂, —CSNH₂, —CONHOH, —CSNHOH, —NHCHO, —NHCONH₂,—NHCSNH₂, —NHSO₃NH2 and —SO₂NH₂; acyl; or —(CH₂CH₂O)_(s)—H, wherein s=1,2, or 3;

or R₁₁ is CH═CH—(CH₂)_(n′)—Q₁₃ or —(CH₂)_(n′)—Q₁₃, wherein Q₁₃ is anaryl or a heteroaryl group substituted with —OH, —COOH, —F, —Cl, —CONH₂,—CSNH₂, —CONHOH, —CSNHOH, —NHCHO, —NHCONH₂, —NHCSNH₂, —NHSO₃NH₂ and—SO₂NH₂, and wherein n′≧0;

R₁₂ and R₁₃ are independently OH, H, F, Cl, OW₁₁, or O(CO)W₁₁, whereinW₁₁ is C₁₋₂₄ alkyl, C₂₋₂₄ alkenyl or C₂₋₂₄ alkynyl, or an aryl or aheteroaryl group substituted with —OH, —COOH, —F, —Cl, —CONH₂, —CSNH₂,—CONHOH, —CSNHOH, —NHCHO, —NHCONH₂, —NHCSNH₂, —NHSO₃NH₂ and —SO₂NH₂;

Z₁₂ is a bond, O, S, or CH₂;

R₁₄ is —(CH₂)_(n″)—Q₁₄, wherein Q₁₄ is an aryl group or a heteroarylgroup substituted with —OH, —COOH, —F, —Cl, —CONH₂, —CSNH₂, —CONHOH,—CSNHOH, —NHCHO, —NHCONH₂, —NHCSNH₂, —NHSO₃NH₂ and —SO₂NH₂, and whereinn″=0, 1, 2, or 3;

Z₁₃ is a bond, O, CH₂, S, SO, SO₂, NQ₁₄Q₁₅, wherein Q₁₄ is H, C₁₋₂₄ acylor C₁₋₂₄ alkyl, and Q₁₅ is CO or —C(O)W₁₂, wherein W₁₂ is O or NW₁₃,wherein W₁₃ is H, OH, C₁₋₂₄ acyl or C₁₋₂₄ alkyl;

R₁₅ is H; C₁₋₂₄ alkyl, C₂₋₂₄ alkenyl or C₂₋₂₄ alkynyl, which alkyl,alkenyl and alkynyl may be substituted with one or more substituentsindependently selected from, —OH, —COOH, —F, —Cl, —CONH₂, —CSNH₂,—CONHOH, —CSNHOH, —NHCHO, —NHCONH₂, —NHCSNH₂, —NHSO₃NH₂ and —SO₂NH₂;acyl or —(CH₂CH₂O)_(s)—H, wherein s=1, 2, or 3;

or R₁₅ is CH═CH—(CH₂)_(n′)—Q₁₃, or —(CH₂)_(n′)—Q₁₃, wherein Q₃ is asdefined above and wherein n′≧0;

or a salt of such a compound.

In the present context, the terms “C₁₋₅, C₁₋₆ and C₁₋₂₄ alkyl” isintended to mean alkyl groups with 1-5, 1-6 and 1-24 carbon atoms,respectively, which may be straight or branched or cyclic such asmethyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert.butyl, hexyl,octyl, dodecyl, cyclopentyl, cyclohexyl, etc.

Further, as used herein, the terms “C₂₋₅, C₂₋₆ and C₂₋₂₄ alkenyl” isintended to mean mono- or polyunsaturated alkyl; groups with 2-5 and2-24 carbon atoms, respectively, which may be straight or branched orcyclic in which the double bond(s) may be present anywhere in the chainor the ring, for example vinyl, 1-propenyl, 2-propenyl, hexenyl,decenyl, 1,3-heptadienyl, cyclohexenyl etc. Some of the substituentsexist both in a cis and a trans configuration. The scope of thisinventions comprises both the cis and trans forms.

In the present context, the terms “C₂₋₅, C₂₋₆ and C₂₋₂₄ alkynyl” isintended to mean a straight or branched alkyl group with 2-5 and 2-24carbon atoms, respectively, and incorporating one or more triplebond(s), e.g. ethynyl, 1-propynyl, 2-propynyl, 2-butynyl,1,6-heptadiynyl, etc.

The terms “C₁₋₆ and C₁₋₂₄ alkoxy” designate alkyl groups as definedabove comprising an oxy function.

In the present context, the term “aryl” is intended to mean phenyl andnaphthyl. The term “heteroaryl” is intended to mean a cyclic aromaticsystem, wherein at least one non-carbon atom contributes to the πbonding system.

Examples of substituted aryl groups are: 3-nitrophenyl, 3-hydroxyphenyl,4-hydroxyphenyl, 3,4-dihydroxyphenyl, 3-carboxamidophenyl,3-formamidylphenyl, 3-acetamidylphenyl, 3-fluoronaptht-2-yl,7-fluoronaphthyl, 3,7-difluoronaphthyl, 3-hydroxynaphthyl,7-hydronaphthyl, 3,7-dihydroxynaphthyl, 3-fluoro-7-hydroxynaphthyl,7-fluoro-3-hydroxynaphthyl, 4-fluoronaphth-2-yl, 6-fluoronaphth-yl,8-fluoronaphth-2-yl, 4,6-difluoronaphth-2-yl etc.

Examples of heterocyclic and heteroaryl groups are pyrrolyl, furanyl,2,3-dihydrofuranyl, tetrahydrofuranyl, thienyl, 2,3-dihydrothienyl,tetrahydrothienyl, imidazolyl, oxazolyl, thiazolyl, pyrazolyl, indolyl,pyrazinyl, dioxolanyl, dioxanyl, 1,3,5-trioxanyl, tetrahydrothiapyranyl,dithiolanyl, pyrazolidinyl, iminazolidinyl, sym-triazinyl,sym-tetrazinyl, quinazolinyl, pteridinyl, isoindolyl, 1,2,4-triazolyl,1,2,3-triazolyl, benzimidazolyl, indazolyl, benzofuranyl,isobenzofuranyl, benzothiophenyl, thienothiophenyl, isoxazolyl,1,2,5-oxadiazolyl, isothiazolyl, 1,3,4-thiadiazolyl, benzoxazolyl,benzothiazolyl, azaindolyl, oxoindolyl, hydroxyindolyl, N-oxyisoquinolyletc.

In the present context, the term “acyl” (e.g. C₁₋₂₄ acyl) is intended todesignate the acyl residue of a carboxylic acid or a sulphonic acidmoiety comprising a carbonyl or sulphonyl group and an organic moiety.Examples of acyl groups include C₁₋₂₄ alkanoyl (e.g. formyl, acetyl,propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl andhexanoyl), C₁₋₂₄ alkenoyl (e.g. acryloyl, metacryloyl, crotonoyl,isocrotonoyl, 2-pentenoyl, 3-pentenoyl, 2-methylpentenoyl, 3-pentenoyl,3-phenylpropenoyl, 2-phenyl-trans-propenoyl, 2,4-hexadienoyl), C₁₋₂₄alkynoyl (e.g. propyonyl, 2-butynoyl, 3-butynoyl, 2-methyl-3-butynoyl,2,2-dimethylbutynoyl, 2-pentynoyl, 3-pentynoyl, 2-pentyn-4-trans-enoyl),C₁₋₂₄ alkoxycarbonyl (e.g. methoxycarbonyl, ethoxycarbonyl,propoxycarbonyl, butoxycarbonyl and t-butoxycarbonyl), C₁₋₂₄alkenyloxycarbonyl (e.g. cis-2-butenyloxycarbonyl,1-methyl-2-propenyloxycarbonyl, 1,1-dimethyl-2-propenyloxycarbonyl,trans-2-butenyloxycarbonyl), aroyl (e.g. benzoyl), heterocyclylcarbonyl(e.g. 2-furoyl, 3-furoyl, 2-furanoyl, 3-furanoyl,-2-pyrrolcarboxyl,3-pyrrolcarboxyl, 2-thenoyl, 3-thenoyl, 2-indolcarboxyl,3-indolcarboxyl, 1-naphthanoyl and 2-naphthanoyl), etc.

The term “salt” is intended to comprise a salt such as an organic acidaddition salt (e.g. acetate, valerate, salicylate, galacturonate,gluconate, tannate, triflouroacetate, maleate, tartrate,methanesulfonate, benzenesulfonate, formiate, thiocyanate andtoluenesulfonate), an inorganic acid addition salt (e.g. hydrochloride,hydrobromide, hydroiodide, dihydrochloride, dihydrobromide,dihydroiodide, sulphate, hydrogensulphate, halosulphate such asiodosulphate, nitrate, phosphate, and carbonate) or a salt with an aminoacid (e.g. arginine, aspartic acid and glutamic acid) or a metal saltsuch as an alkali metal salt (e.g. sodium salt and potassium salt) andan earth alkali metal salt (e.g calcium salt and magnesium salt), anammonium salt, an organic alkali salt (e.g. trimethylamine salt,triethylamine salt, pyridine salt, picoline salt, dicyclohexylamine saltand N,N′-dibenzylethylenediamine salt), and hydrates thereof.

When the substituent R₅ designates heterocyclyl, it is preferred thatthe substituent designates a heterocyclyl group of the formula

wherein ′X is —CH—, —CH₂—, —O—, —N—, —S—, —S→O, —N→O or —CO—, Y is —CH—or —NH—, and Z is —CH—, —CH₂—, —O—, —S—, —N—, —CO—, —S→O or —N→O,especially a group selected from the group consisting of inden-7-yl,benzofuran-4-yl, isobenzofuran-4-yl, thionaphthen-4-yl,isothionaphthen-4-yl, 2-oxo-inden-7-yl, 2-oxo-inden-4-yl, inden-4-yl,benzofuran-7-yl, isobenzofuran-7-yl, thionaphten-7-yl,isothionaphthen-7-yl, 1-oxo-thionaphthen-4-yl, 1-oxo-thionaphthen-7-yl,anthran-4-yl, anthran-7-yl, thioanthran-4-yl, thioanthran-7-yl,benzthiozol-4-yl, benzthiozol-7-yl, 2H-2-isobenzo-1,3-dion-7-yl,isobenzofuran-5-yl, isobenzofuran-6-yl, 3H-2-oxo-benzofuran-5-yl,3H-2-oxo-benzofuran-6-yl, 3H-2-oxothionaphthen-5-yl,3H-2-oxothionaphthen-6-yl, indol-5-yl, indol-6-yl, 3H-2-oxoindol-5-yl,3H-2-oxoindol-6-yl, 3H-2-oxobenzoxazol-5-yl, 3H-2-oxobenzoxazol-6-yl,benzothiazol-5-yl, benzothiazol-6-yl, 2-oxobenzo-1,3-dithiol-5-yl,2-oxobenzo-1,3-dithiol-6-yl, 3H-2-oxobenzimidazol-5-yl,3H-2-oxobenzimidazol-6-yl, benzoxathiol-5-yl, benzoxathiol-6-yl,3H-2-oxobenzthiazol-5-yl and 3H-2-oxobenzthiozol-6-yl.

It is also preferred that the substituent R₅ is a group of the formula

or that R₅ is a group of the formula

With respect to the substituent R₂ being heterocyclyl, it is especiallypreferred that it is selected from the group consisting ofisobenzofuran-5-yl, isobenzofuran-6-yl, 3H-2-oxo-benzofuran-5-yl,3H-2-oxo-benzofuran-6-yl, 3H-2-oxothionaphthen-5-yl,3H-2-oxothionaphthen-6-yl, indol-5-yl, indol-6-yl, 3H-2-oxoindol-5-yl,3H-2-oxoindol-6-yl, 3H-2-oxobenzoxazol-5-yl, 3H-2-oxobenzoxazol-6-yl,benzothiazol-5-yl, benzothiazol-6-yl, 2-oxobenzo-1,3-dithiol-5-yl,2-oxobenzo-1,3-dithiol-6-yl, 3H-2-oxobenzimidazol-5-yl,3H-2-oxobenzimidazol-6-yl, benzoxathiol-5-yl, benzoxathiol-6-yl,3H-2-oxobenzthiazol-5-yl and 3H-2-oxobenzthiozol-6-yl.

The exact number of substituents present on an alkyl, alkenyl or alkynylmoiety R₁ will be dependent on the length of the carbon chain, thepurpose of the substituents being to cause the entire group R₁ to becompatible with water since, in the chaperone-ligand-complex such as thePapD-ligand complex, the group R₁ will extend into the surroundingaqueous medium. Thus, for a fairly short carbon chain such as up to fourcarbon atoms, it is contemplated that one of the above polarsubstituents will be sufficient, in particular when the substituent islocated terminally whereas, for longer chains, a larger number ofsubstituents, such as a substituent for every other carbon atom, may berequired.

The 4,6-O-(4′-Methoxy)phenylmethylidene-α-D-glucohexopyranoside or4,6-O-(4′-Methoxy)phenylmethylidene-β-D-glucohexopyranoside glycosides:

(used here as preferred examples, but other arylmethylidene orvinylidene acetals may be used) can be prepared as follows: Peracylatedglucose is reacted with, e.g. hydrogen bromide or hydrogen chloride in asuitable solvent such as, e.g. acetic acid or dichloromethane, to formper-O-acylated glycosyl bromide or chloride (O-acylation and glycosylhalide synthesis: M. L. Wolfrom and A. Thompson, Methods in CarbohydrateChemistry, Vol. 2, 211-215, ed. by R. L. Whistler and L. Wolfrom,Academic Press, New York, 1963; G. Hewit and G. Fletcher Jr., ibid,226-228; and R. U. Lemieux, ibid, 223-224)

The suitably protected, when necessary, aglycone alcohol or thiol(H-W₁R₁-PG₁ or H-W₁R₁) (protecting groups: Protective Groups in OrganicSynthesis, Editors T. W. Greene and P. G. M. Wuts, John Wiley & Sons,Inc., New York, 1991) is reacted with the per-O-acylated glucose using aLewis acid such as borontrifluoride etherate (R. J. Ferrier and R. H.Furneaux, Carbohydr. Res., 52 (1976) 63-68; J. Dahmén, T. Frejd, G.Grönberg, T. Lave, G. Magnusson, and G. Noori, Carbohydr. Res 116 (1983)303-307) or trimethylsilyltrifluoromethanesulphonate (T. Ogawa, K.Beppu, S. Nakabayashi, Carbohydr. Res., 93 (1981) C₆-C₉) as promoters.The reaction is carried out in a suitable solvent such as chloroform,dichorometane, or toluene. When the monosaccharide derivative inquestion is a per-O-acylated glycosyl bromide or chloride, promotorssuch as silver trifluoromethane sulphonate or mercury (II)salts (H.Paulsen, Angew. Chem. Int. Ed. Engl., 21 (1982) 155-173) can be used andthe reactions are carried out in a suitable solvent such, asdichlorometane or toluene. The glucose W₁R₁ or W₁R₁PG₁-glycosides areobtained after de-O-acylation using sodium methoxide (A. Thompson, M. L.Wolfrom, and E. Pascu, page 215-220, Methods in Carbohydrate Chemistry,Vol II, Editors: R. L. Whistler and M. L., E. Wolfrom, Academic Press,New York, 1963) in methanol or in methanol containing a co-solvent suchas dichlormethane or tetrahydrofurane.

The 4,6-(4′-methoxy)benzylidene acetals are then obtained by reactionwith 4-methoxybenzaldehyde dimethyl acetal and acid in a polarnon-protic solvent such as e.g. dimethyl formamide, acetonitrile ortetrahydrofurane (J. J. Patroni et al., Aust. J. Chem. 1988,(41),91-102;for other methods of acetal formation, see for example A. N. de Belder,1979, adv. Carbohydr. Chem. Biochem., 34, 179 and references citedtherein).

The epoxides B1 or B2

are then obtained through sulphonate esters: The manno epoxides B₁ canbe prepared by reacting the glucoside derivative A with sodium hydrideand p-toluenesulehonylimidazol in dimethylformamide (D. Hicks and E.Fraser-Reid; Synthesis 1974, 203) or with sodium hydride andp-toluenesulahonyl chloride in tetrahydrofurane (V. S. Murthy et al,Synthetic Commun. 1993, 23(3), 285-289).

The allo epoxides B2 can be prepared by reacting the glucosidederivative A with methylsulphonyl or p-toluenesulphonyl chloride inpyridine and treating the resulting methylsulphonate diester with sodiumethoxide in ethanol (Y. Ali, A. C. Richardson, Carbohydrate Res. 1967,5, 441-448; N. Richtmeyer, Methods in Carbohydrate Chemistry, Vol1,107).

The epoxides B1 or B2 can be reacted with suitable nucleophilic reagentsto yield the diaxially substituted allo hexopyranosides C1 and C₂

(for general references on the use of epoxides, see e.g. J. GorzynskiSmith, Synthesis, 1984, 6, 629-656 Masamune S., Choy W., Petersen J S,and Sita L R, Angew. Chem. Int. Ed. Engl., 1985, 24, 1-76; A. S. Rao etal., Tetrahedron Lett., 1983, 39, 2323).

When the first atom of R₂ and Z₁ (as defined above) connected to thecarbohydrate moiety in the desired final product is a nitrogen (=thenucleophilic atom), then the preferred nucleophile Nu₁ or Nu₂ is azide(N₃ ⁻). The epoxide is treated with sodium azide and ammonium chloridein boiling 2-methoxy-ethanol (R. D. Guthrie and D. Murphy; J.Chem.Soc.1963, 5288-5294).

When the nucleophilic atom is oxygen or sulphur, the preferred generalmethod of epoxide opening involves treatment with suitably protectedalcohol or thiol in the presence of neutral alumina in ether (G. H.Posner and D. Z. Rogers, J. Am. Chem. Soc. 1977, 99, 8208; G. H. Posner,D. Z. Rogers and A. Romero, Isr. J. Chem. 1979, 18, 259; and G. H.Posner, M. Hulce and R. K. Rose, Synth. Commun. 1981, 11, 737).

When the nucleophilic atom is carbon, the most commonly used reagentsare organomagnesium, organolithium, organocopper, organoaluminium andorganoboron compounds (J. Gorzynski Smith, Synthesis, 1984, 8, 629-656and references cited therein).

When the product is an allo hexopyranoside C1, the 2-hydroxy functioncan either be blocked with a protective group that allows for theintroduction of the R₂ functionality at a later stage (preferred if R₂is an ester, not shown in figure), or the suitably protected, whennecessary, functionality R2 is introduced to produce D1

For example, OH-groups to ethers or esters (Protective Groups in OrganicSynthesis, Editors T. W. Greene and P. G. M. Wuts, John Wiley & Sons,Inc., New York, 1991); OH-groups to carbonates (J. March, AdvancedOrganic Chemistry-Reaction Mechanisms, and Structure, 3rd Edn. JohnWiley & Sons, New York, 347 (1985), and references cited therein);OH-groups to carbamates (J. March, Advanced Organic Chemistry-ReactionMechanisms, and Structure, 3rd Edn. John Wiley & Sons, New York, 791-792(1985), and references cited therein); OH-groups to alkylgroups viaexomethylene derivatives and subsequent hydrogenation or via otherroutes (H. O. H. House, Modern Synthetic Reactions, 2nd Edn. W. ABenjamin, Inc., Menlo Park, Calif. 1-130 (1972), and references citedtherein; J. Yoshimura, Adv. Carbohydr. Chem Biochem., 42 (1984) 69-134);and exchange of OH-groups for heterocyclic groups, via different routes(A. R. Katritzky, Handbook of Heterocyclic Chemistry, Pergamon Press,Oxford, 1985).

When the product is an allo hexopyranoside C2, the 3-hydroxy function isblocked with a protective group that allows for the introduction of theZ₁-R₃ functionality at a later stage resulting in intermediates of thetype D2

(Protective Groups in Organic Synthesis, Editors T. W. Greene and P. G.M. Wuts, John Wiley & Sons, Inc., New York, 1991) or transformed to amanno hexopyranoside intermediate D3

where Nu₁ is a protected or masked form of the functionality Z₁.OH-groups to ethers or esters (Protective Groups in Organic Synthesis,Editors T. W. Greene and P. G. M. Wuts, John Wiley & Sons, Inc., NewYork, 1991); OH-groups to azido-groups: J. March, Advanced OrganicChemistry-Reaction Mechanisms, and Structure, 3rd Edn. John Wiley &Sons, New York, 380, (1985), and references cited therein; H. H. Baer,Pure Appl. Chem., 61(7) (1989) 1217-1234, and references cited therein;OH-groups to aminogroups via azides or other routes (March, AdvancedOrganic Chemistry-Reaction Mechanisms, and Structure, 3rd Edn. JohnWiley & Sons, New York, 1106, 798-800 (1985), and references citedtherein; H. H. Baer, Pure Appl. Chem., 61(7) (1989) 1217-1234, andreferences cited therein).

The 4,6-O-acetal function is then reductively opened to yield either thefunction R₆ in the case when R₆ is an ether, or the intermediates F1, F2or F3 with a hydroxy function on position 6 (reductive opening ofacetals, see Garegg P J and Hultberg H, Carbohydr. Res. 1981, 93,c10-11; Garegg P J, Hultberg H, and Wallin S, Carbohydr. Res. 1982, 108,97-101; Liptak A, Jodal I, Nanasi P, Carbobydr. Res. 1975, 44, 1-11;Baker D C, Horton D, Tindall C G, Methods in Carbohydr. Chem., 1976 Vol6, 3-6; Mikami T, Asano H, Mitsunobu O, Chem. Lett. 1987, 10, 2033-2036;Ek M, Garegg P J, Hultberg H, Oscarsson S, J. Carbohydr. Chem. 1983, 2,305-311; Hunter R, Bartels B, Michael J P, Tetrahedron Lett. 1991, 32,1095-1098; Rao S P, Grindley T B, Carbohydr. Res. 1991, 218, 83-93;Hunter,R. Bartels,B. J. Chem. Soc. Chem. Commun. 1991, 2887-2888).

For example, the intermediates of type D1, D2 or D3 are treated withsodium cyanoborohydride and chlorotrimethylsilane in acetonitrile, (R.Johansson and B. Samuelsson, J. Chem. Soc. Perkin Trans. 1, 1984,2371-2374) or borane-trimethylamine complex and aluminium trichloride.The regiochemical outcome of the reaction is often solvent-dependent (EkM, Garegg P J, Hultberg H, Oscarsson S, J. Carbohydr. Chem. 1983, 2,305-311).

The aldehyde intermediates of type G1, G2 or G3

are obtained by oxidation of the corresponding 6-alcohol intermediatesof type D1, D2 or D3, preferrably by the Swern procedure. (Mancuso A J,Swern D, Synthesis, 1981, 165-185; Tidwell T, Synthesis, 1990, 857-870;for other oxidation methods, see A. H. Haines, 1988, Methods for theOxidation of Organic Compounds, Chapter 2, Academic Press, San Diego,and references cited therein).

In the next step a carbon nucleophile is added to the aldehyde functionof the intermediates of type G1, G2 or G3. Preferrably, a suitablyprotected alkyllithium or aryllithium reagent or a gringard reagent isadded to the aldehyde in an ether or hydrocarbon solvent to produce thesecondary alcohols H1, H2 and H3:

For reactions of aldehydes with organolithium and organomagnesiumcompounds, see J. March, Advanced Organic Chemistry-Reaction Mechanisms,and Structure, 3rd Edn. John Wiley & Sons, New York, 347 (1985), andreferences cited therein.

For reactions of aldehydes with organolithium and organomagnesium andother carbon nucleophils, see Evans D A, Aidrichim. Acta, 1982, 15, 23,and references cited therein.

For specific examples on the use and preparation of arylsubstitutedphenyllithium and grignard reagents, see Ames M M, Castagnoli Jr. N, J.Labelled Compd., 1974, 10(2), 195-205; DE U.S. Pat. No. 3,807,910 A1;Mills R J, Snieckus V, Polynucl. Aromat. Hydrocarbons, [Pap. Int.Symp.], 8th, Meeting 1983, 913-24. Edited by: Cooke M and Dennis A J,Battelle Press 1985: Columbus, Ohio; Iriye R, Furukawa K, Nishida R, KimC, Fukami H, Biosci. Biotechnol. Biochem. 1992, 56(11), 1773-5; Comber MF, Sargent M V U, J. Chem. Soc., Chem. Commun., 1991, (3), 190-2; HiraiT, Yoshizawa A, Nishiyama I, Fukumasa M, Shirátori . N, Yokoyama A, EP341686 A2; Leeson P D, Emmett J C, Shah V P, Showell G A, Novelli R,Prain H D, Benson M G, Ellis D, Pearce N J, Underwood A H, J. Med.Chem.1989, 32(2), 320-36); Meltzer P C, Liang A Y, Brownell A L, ElmalehD R, Madras B K, J. Med. Chem., 36(7), 855-62; Willard A K, Novello F C,Hoffman W F, Cragoe Jr E J. U.S. Pat. No. 4,459,423.

For substituted phenyllithium reagents that can be further elaboratedinto heterocyclic compounds, see: Lang H J,; Muschaweck R, AU 514406 B2;Lang H J, Muschaweck R, Hropot M, HU 19761; Lang H J, Muschaweck R,Hropot M, DE U.S. Pat. No. 2,737,195.

For heteroaromatic aryllithiums and grignard reagents, see: Yang Y,Martin A R, Nelson D L, Regan J, Heterocycles, 1992, 34(6), 1169-75.

For examples of other organometallic reagents for the stereo-selectivesynthesis of secondary alcohols from aldehydes:

Homoallyl alcohols with crotylmolybdenum complexes: Faller J W. John JA, Mazzieri M R, Tetrahedron Lett. 1989, 30, 1769-1772.

Homoallyl alcohols with titanium complexes: Riediker M, Duthaler R O,Angew. Chem. Int. Ed. Engl., 1989, 28, 494-495.

3-Pyrollyl alcohols with silyl protected 3-lithiopyrrole: Bray B L,Mathies P H, Naef R, Solas D R, Tidwell T T, Artis D R, Muchowski J M,J. Org. Chem., 1990, 55, 6317-6328.

Allylic alcohols with E-vinylalane: A P Kozikowski and Jiang-Ping Wu,Tetrahedron Lett. 1990, 30, 4309-4312 and references cited therein.

Trans-allylic diols with vinylstannanes: Corey E J, Wollenberg R H, J.Org. Chem., 1975, 40, 2265-2266.

Pyrrolidine carbinols with α-lithio pyrrolidine amidines: Sanner M A,Tetrahedron Lett. 1989, 30, 1909-1912.

Organozinc reagents: Fürstner A, Synthesis, 1989, 571-590, andreferences cited therein.

Thereafter, the substituent on the 3-position (Nu₁ or OPG₁) istransformed into a nucleophile in order to install the negativelycharged functionality Z₁-D-R₃.

The secondary 6-alcohols H1, H2 and H3 are first protected, (ProtectiveGroups in Organic Synthesis, Editors T. W. Greene and P. G. M. Wuts,John Wiley & Sons, Inc., New York, 1991) or transformed to aminofunctions in the cases where X₁-R₆ forms a peptide functionality(OH-groups to aminogroups via azides or other routes: See for examplesMarch, Advanced Organic Chemistry-Reaction Mechanisms, and Structure,3rd Edn. John Wiley & Sons, New York, 1106, 798-800 (1985), andreferences cited therein; H. H. Baer, Pure Appl. Chem., 61(7) (1989)1217-1234, and references cited therein).

For peptide synthesis, see Gross and Meienhofer, The Peptides, 3 vol.,Academic Press, New York,1979-1981; Grant G A et al., SyntheticPeptides, A Users Guide, 1992, W. A. Freeman and Company, New York, andreferences cited therein).

The 3-position is deprotected to an alcohol, thiol or amine intermediateI1, I2 or I3

(Protective Groups in Organic Synthesis, Editors T. W. Greene and P. G.M. Wuts, John Wiley & Sons, Inc., New York, 1991, and references citedtherein).

For example, treatment of I1, where R₂-PG₁ is a combination of etherfunctions and Q₁ is azide, with gaseous hydrogen sulphide in pyridineand water (T. Adashi, Y. Yamada, I. Inoue and M. Saneyoshi, Synthesis,1977, 45). For other methods of azide reductions, see Poopeiko N E,Pricota T I, Mikhailopulo I A, Synlett, 1991, 5, 342; Samano M C, RobinsM J, Tetrahedron Lett. 1991, 32, 6293-6296; Rakotomanomana N, LacombeJ-M, Pavia A, Carbohydr. Res., 1990, 197, 318-323; Malik A A, Preston SB, Archibald T G, Cohen M P, Baum K, Synthesis, 1989,450-451; Maiti S,Spevak P, Reddy N, Synt. Commun. 1988, 18, 1201-1206; Bayley H,Standring D N, Knowles J R, Tetrahedron Lett., 1978, 39, 3633-3634).

The intermediates I1, I2 or I3 are sulphated with sulphurtrioxide-pyridine or -triethylamine complex to obtain the intermediatesJ2, J4 and J6

(see, for example J. Basten, G. Jaurand, B. Olde-Hanter, M. Petitou andC.A.A. van Boeckel, Bioorg. Med. Chem. Lett, 1992, 2(9), 901-904 andreferences cited therein; J.Basten, G. Jaurand, B. Olde-Hanter, P.Duchaussoy, M. Petitou and C. A. A. van Boeckel, Bioorg. Med. Chem.Lett, 1992, 2(9), 905-910, and references cited therein; Böcker, T.Lindhorst, T K. Thiem, J. Vill, V. Carbohydr. Res. 1992, 230, 245-256)or coupled to form the phosphoester intermediates J1, J3 and J5

Since the phosphorus-nitrogen bond is known to be acid labile, theintermediates leading to phosphodiester end-products are preferred (M.Selim and T. N. Thanh, C.R. Seances Acad. Sci, 1960, 250, 2377).

For example, the alcohol intermediates I1, I2 or I3 are treated with2,2,2-trichloroethyl 2-chlorophenyl phosphochloridate in chloroform andpyridine to form the phosphate triesters. The 2,2,2-trichloroethyl groupis removed by treatment with zinc powder and the resulting phosphatediester is activated with3-nitro-1-(2,4,6-triisopropylbenzenesulphonyl)-1,2,4-triazol e andcoupled with the alcohol R₃-OH to form the intermediates J1, J3 and J5.The 2-chlorophenyl group is removed by treatment withpyridine-2-aldoxime and N,N,N,N-tetramethylguanidine in moist pyridine(see for example P. J. Garegg, R. Johansson, I. Lindh and B. Samuelsson,Carbohydr. Res. 1986, 150, 285-289).

Alternatively, by the phosphite triester approach, alcohol intermediatesI1, I2 or I3 are treated with phenylchloro-N,N-diisopropylphosphoramidite in acetonitrile to formcarbohydrate phosphoamidites. After purification by chromatography,these are exposed to an alcohol R₃—OH in the presence of a mild acid,such as pyridinium p-toluenesulphonate and treated with tert-butylhydroperoxide to form phosphate diesters J1, J3 and J5 (PG₃ =H) (H-N.Caro, M. Martín-Lomas and M. Bernabé, Carbohydr. Res. 1993, 240,119-131, and references cited therein).

For a review on phospodiesters in DNA synthesis, see Narang S,Tetrahedron, 1983, 39, 1-22 and D. W. Hutchinson, 1991, Chapter 3 inChemistry of Nucleosides and Nucleotides, vol 2, ed. B. Townsend, PlenumPress, New York, and references cited therein. For other examples oncarbohydrate phosphodiester synthesis, see for example Ichikawa Y, Sim MM, Wong C H, J. Org. Chem. 1992, 57, 2943-2946 and Schmidt R R, Braun H,Jung K-H, Tetrahedron Lett. 1992, 33, 1585-1588. For synthesis ofmodified phosphodiester linkages, See R. S. Varma, 1993, Synlett,621-636, and references cited therein.

To obtain arylphosphonic acid esters and amides J1, J3 and J5, where R₃is an alkyl or aromatic group the arylphosphonic acid R₃-PO₃H₂ iscoupled to the alcohol intermediates I1, I2 or I3, for example, with acarbodiimide reagent, or treatment of the phosphonic dichlorides withthe alcohol intermediates I1, I2 or I3 in pyridine (T. H. Siddal III,and C. A. Prohaska, J. Am. Chem. 1962, 84, 3467).

The alkylphosphonic triesters are formed from trialkyphosphites andalkyl halides by the Arbuzov reaction (Arbuzov, Pure Appl. Chem. 1964,9, 307-335, J. March, Advanced Organic Chemistry-Reaction Mechanisms,and Structure, 3rd Edn. John Wiley & Sons, New York, 347 (1985), andreferences cited therein).

For the preparation of arylphosphonic triesters via phosphorustrichloride, see for example: K. Sasse, Methoden der Organichen Chemie(Houben-Weyl), 4th ed., Vol. 12/1, Georg Thieme, Stuttgart, 1963, p. 314and p. 392 and references cited therein; G. M. Kosolapoff, Org. React.6, 273 (1951), and references cited therein; L. D. Freedman and G. O.Doak, Chem. Rev. 1957, 57, 479, and references cited therein.

For the preparation of arylphosphonic triesters via organomagnesium ororganolithium reagents, see for example: K. Sasse, Methoden derOrganichen Chemie (Houben-Weyl), 4th ed., Vol. 12/1, Georg Thieme,Stuttgart, 1963, p. 372, and references cited therein; G. M. Kosolapoff,Org. React. 6, 273 (1951), and references cited therein.

Intermediates J1, J2, J3, J4, J5 and J6 are deprotected to form thefinal products (Protective Groups in organic Synthesis, Editors T. W.Greene and P. G. M. Wuts, John Wiley & Sons, Inc., New York, 1991, andreferences cited therein) and transformed to their sodium or potassiumsalts.

It is justified to assume that the compounds described above are capableof interacting with sites in PapD and other periplasmic chaperones. Inorder to establish that this is really the case, assays like thosedescribed in the examples should be performed.

Thus, a preferred compound of the invention is a compound as describedabove, which causes a prevention, inhibition or enhancement of thebinding of G1′-19′WT to PapD, and/or causes a prevention, inhibition orenhancement of the binding of the fusion peptide MBP-G1′-140′ to PapDand/or causes a prevention, inhibition or enhancement of the binding ofthe peptide G125′-140′ (which have the sequence SEQ ID NO: 20) to PapDand/or is capable of inhibiting the restoration of the PapD-PapG complexnormally caused by the addition of access PapD.

The assay used to establish that a substance exhibits one of the aboveeffects is preferably one of the assays described in the examplesherein. Of course, also. other assays as those discussed above in themethods of the invention may be utilized in order to establish that thecompound actually is capable of inhibiting pilus assembly.

One important point which should be taken into consideration whensetting up an assay, is the role the chaperones are playing in vivo.They bind to the pilus subunits already during the transport through thecell membrane of the subunits and it is therefore assumed, that thepilus subunits are more or less unfolded (i.e. not in their final foldedconformation) when they bind to the chaperone. It has been observed bythe inventors that the kinetics of binding between completely foldedpilus subunits (or analogues thereof) and the chaperone PapD is a slowprocess, although it is known that the process of pilus assembly isrelatively fast in vivo. In order to speed up the rate of assembly ofthe chaperone/pilus subunit complex in vitro it is contemplated thatmore or less severe denaturing conditions could be imposed on the pilussubunits (or the analogues thereof) prior to the assay. Such denaturingcould be obtained by subjecting the pilus subunits to physical stress(e.g. to elevated temperature, pressure changes, radiation etc.) or tochanges in the chemical environment (e.g. changes in ionic strength,changes in pH, or the addition of denaturing compounds or disulphidereducing compounds.

The expression “denaturing compound” refers to a compound which whenpresent as one of the solutes in a liquid phase comprising polypeptidemolecules may destabilize folded states of the polypeptide moleculesleading to partial or complete unfolding of the polypeptide chains. Thedenaturing effect exerted by a denaturing compound increases withincreasing concentration of the denaturing compound in the solution, butmay furthermore be enhanced or moderated due to the presence of othersolutes in the solution, or by changes in physical parameters, e.g.temperature or pressure.

As examples of suitable denaturing compounds to be used may be mentionedurea, guanidine-HCl, di-C₁₋₆alkylformamides such as dimethylformamideand di-C₁₋₆-alkylsulphones.

Examples of disulphide reducing compounds are glutathionyl-2-thiopyridyldisulphide, 2-thiocholyl-2-thiopyridyl disulphide,2-mercaptoethanol-2-thiopyridyl disulphide andmercaptoacetate-2-thiopyridyl disulphide.

A set of observations confirm the assumption that the pilus subunits aremore or less unfolded while bound to the chaperone: As described inKuehn et al., 1991, it is possible to restore the PapD-PapG complexafter denaturation by adding access PapD. Further, preliminary resultsobtained by capillary electrophoresis show that denaturation of thefusion protein MBP-G1′-140′ gives a single form of the fusion protein,whereas two forms of the fusion protein are observed in capillaryelectrophoresis before denaturation, one major which is unable tointeract with PapD and one minor which is capable of interacting withPapD. It is therefore contemplated that the denatured fusion protein mayserve as a superior substrate in the different competitive assaysdescribed herein as does the non-denatured form of the fusion protein.

Thus, in a preferred embodiment of the invention, the compound of theinvention causes a prevention, inhibition or enhancement of the bindingof a denatured form of either a pilus subunit or an analogue thereof toPapD, and/or causes a prevention, inhibition or enhancement of thebinding of a denatured form of MBP-G1′-140′ to PapD and/or causes aprevention, inhibition or enhancement of the binding of a denatured formof G125′-140′ to PapD.

As will appear from the examples, compounds which should be capable ofinteracting with PapD and other chaperones have already been identifiedand synthesized. These compounds which are all pyranosides are also animportant part of the invention:

Ethyl 2,3-O-Dibenzoyl-4-O-benzyl-1-thio-β-D-glucohexopyranoside;

Ethyl6-O-acetyl-2,3-O-dibenzoyl-4-O-benzyl-1-thio-β-D-glucohexopyranoside;

Methylglycolyl6-O-acetyl-2,3-O-dibenzoyl-4-O-benzyl-β-D-glucohexopyranoside;

2-(Hydroxy)ethyl 4-O-benzyl-β-D-glucopyranoside;

Sodium glycolyl 4-O-benzyl-β-D-glucohexopyranoside;

Methyl2-O-ethyl-4,6-O-(4′-methoxy)phenylmethylene-α-D-mannohexopyranoside;

Methyl2-O-ethyl-3-O-dimethyl-t-butylsilyl-4,6-O-(4′-methoxy)phenylmethylene-α-D-mannohexopyranoside;

Methyl2-O-ethyl-3-O-dimethyl-t-butylsilyl-4,-O-(4′-methoxy)benzyl-α-D-mannohexopyranoside;

methyl2-O-ethyl-3-O-dimethyl-t-butylsilyl-6-O-(4′-methoxy)benzyl-α-D-mannohexopyranoside;

Methyl2-O-ethyl-3-O-dimethyl-t-butylsilyl-4,-O-(4′-methoxy)benzyl-6(S)-phenyl-α-D-mannohexopyranoside;

Methyl 2,3-anhydro-4,6-O-p-methoxybenzylidene-α-D-mannopyranoside;

Methyl 3-azido-4,6-O-p-methoxybenzylidene-α-D-altropyranoside;

Methyl 3-azido-2-O-ethyl-4,6-O-p-methoxybenzylidene-α-D-altropyranoside;

Methyl 3-azido-3-deoxy-2-O-ethyl-4-O-p-methoxybenzyl-α-D-altropyranosid;

Methyl3-azido-6-O-benzoyl-3-deoxy-2-O-ethyl-4-O-p-methoxy-benzyl-α-D-altropyranoside;

Methyl6-O-benzoyl-3-deoxy-2-O-ethyl-4-O-p-methoxybenzyl-3-sulfamino-α-D-altropyranosidesodium salt;

Methyl 6-O-benzoyl-3-deoxy-2-O-ethyl-3-sulfamino-α-D-altropyranosideammonium salt;

Methyl3-azido-6-O-pivaloyl-3-deoxy-2-O-ethyl-4-O-p-methoxy-benzyl-α-D-altropyranoside;

Methyl6-O-pivaloyl-3-deoxy-2-O-ethyl-4-O-p-methoxybenzyl-3-sulfamino-α-D-altropyranosidesodium salt;

Methyl 6-O-pivaloyl-3-deoxy-2-O-ethyl-3-sulfamino-α-D-altropyranosideammonium salt;

Methyl6-O-pivaloyl-3-deoxy-2-O-ethyl-4-O-p-methoxybenzyl-3-tbutyloxamido-α-D-altropyranoside;

Methyl 6-O-pivaloyl-3-deoxy-2-O-ethyl-3-oxamido-α-D-altropyranosideammonium salt;

Methyl3-azido-6-O-pyrrol-3′-ylcarboxyl-3-deoxy-2-O-ethyl-4-O-p-methoxybenzyl-α-D-altropyranoside;and

Methyl6-O-pyrrol-3′-ylcarboxyl-3-deoxy-2-O-ethyl-3-sulfamino-α-D-altropyranosideammonium salt.

Other compounds are of course also possible as interactors with sites inchaperones. As is evident from example 7, modified peptides also mayprove to be useful in the methods of the invention.

As will be clear from the above, the identification of a site in thechaperone which may be affected so as to interfere with pilus assemblyis a critical starting point in the methods described herein for theidentification, isolation and synthesis of compounds capable ofinteracting with periplasmic chaperones.

Thus, the invention also relates to a method for identifying a bindingsite in a molecular chaperone, comprising

co-crystallizing the periplasmic molecular chaperone or an analoguethereof with a ligand binding to the periplasmic molecular chaperone orthe analogue thereof,

resolving the three-dimensional structure of the chaperone/ligandinteraction, thereby resolving the three-dimensional structure of theperiplasmic molecular chaperone or the analogue thereof when binding tothe ligand,

determining the site-point(s) involved in the intermolecular interactionbetween the periplasmic molecular chaperone or the analogue thereof andthe ligand, and

identifying the thus determined site-point(s) of the periplasmicmolecular chaperone or the analogue thereof as a binding site in theperiplasmic molecular chaperone or the analogue thereof.

By the term “ligand” as used herein, is meant a substance which exhibitbinding to a host or receptor molecule (in this connection a chaperone).The binding is not a non-specific interaction, which means that abinding motif between the ligand and the host or receptor moleculeexists. In other words, when bringing a sample of the ligand and asample of the host or receptor molecule in contact with each other thecomplexes formed between the ligand and the host or receptor moleculewill substantially all reflect the same intermolecular interactions.

As mentioned above one embodiment of the invention is to administer asubstance which is capable of preventing, inhibiting or enhancingbinding between pilus subunits and molecular chaperones.

Accordingly the invention relates to a pharmaceutical composition,comprising, as an active compound, a substance capable of interactingwith at least one type of periplasmic molecular chaperone which bindspilus subunits during transport of these pilus subunits through theperiplasmic space and/or during the process of assembly of the intactpilus, in such a manner that binding of pilus subunits to theperiplasmic molecular chaperone is prevented, inhibited or enhanced, incombination with at least one pharmaceutically acceptable carrier orexcipient. Preferably such a substance is a substance according to theinvention or a substance identified/designed according to the methods ofthe invention.

The pharmaceuticals and pharmaceuticals discussed herein are thus,according to the invention, for the treatment and/or prophylaxis of thesame conditions as those discussed when disclosing the method oftreatment of the invention and caused by the same bacterial species.

Therefore, a pharmaceutical composition, comprising, as an activecompound, a substance used in the therapeutic methods of the invention,a substance according to the invention or a substance identifiedaccording to the methods of the invention, in combination with at leastone pharmaceutically acceptable carrier or excipient, is a part of theinvention.

Such pharmaceutical compositions of the invention could also comprise atleast one additional pharmaceutical substance, which i.a. could enhancethe pharmaceutical effects exerted by the substance of the invention orthe substance identified according to the invention.

Additional pharmaceutical substances could be steroid hormones,disinfectants, anti-pyrretics, etc. Preferably such an additionalsubstance could be an antibacterial agent.

Such an anti-bacterial agent could conveniently be selected from thegroup consisting of penicillins, cephalosporins, aminoglycosides,sulfonamides, tetracyclines, chloramphenicol, polymyxins,antimycobacterial drugs and urinary antiseptics.

The invention also relates to a substance employed in the methods of theinvention as well as a substance of the invention and a substanceidentified according to the methods of the invention for use as apharmaceutical. It is preferred that the pharmaceutical is foranti-bacterial treatment and/or prophylaxis, especially treatment and/orprophylaxis of diseases caused by tissue-adhering pilus formingbacteria, and it is especially preferred that the pharmaceutical is fortreatment and/or prophyloxis of urinary tract infection.

Finally, the invention relates to the use of a substance employed in themethods of the invention as well as a substance of the invention and ofa substance identified according to the methods of the invention for thepreparation of a pharmaceutical composition for the treatment and/orprophylaxis of bacterial infection.

LEGENDS TO FIGURES

FIG. 1: PapD binds to pilus subunit-related peptides coated in ELISAmicrotiter plates and in solution.

A and B: The peptides from table 1 were coated on microtiter wells andtested for their ability to bind to PapD in an ELISA assay described inexample 2. Water insoluble peptides were dissolved in 2.5% acetic acid(AA) which had no effect on the binding of G1′-19′WT to PapD in thisassay. Each graph represents the average of duplicate wells.

FIG. 2: Binding of pilus subunit-related peptides provides protectionagainst enzymatic proteolysis and blocks binding of PapD to PapG.

A Upper: PapD (15 μg) was incubated with PBS (lane D) or with 1.5 μgtrypsin (lane D and Tr) at 37° C. for 20 minutes and applied to 20%SDS-PAGE. Coomassie blue stained bands corresponding to full length PapD(PapD), trypsin (Tr, the NH₂-terminal fragment of PapD (N), and theCOOH-terminal fragment of PapD starting at residue 100 (C) areindicated).

A Lower: The rate of trypsin cleavage of PapD decreased uponpreincubation with the G1-19′WT, G1-16′WT or K1′-19′WT peptides, but theG2′-21′amide peptide had no effect. 50 μg of purified PapD waspreincubated for 15 min at 25° C. with a 20-fold molar excess ofG1′-19′WT, G1′-16′WT, K1′-19′WT or G2′-21′amide peptides (any trypsincleavage sites in these peptides occur at their amino termini where theywere predicted not to interfere with PapD), or an equivalent volume ofwater. Each sample was then incubated at 37° C. with 3.2 μg of trypsin.Aliquots were removed after 0, 5, 10, 20, 30 and 40 min and boiled inSDS-PAGE sample buffer to stop the digestion. The samples were appliedto 15% SDS-PAGE gels and the amount of full-length PapD remaining wasdetermined by densitometry of Coomassie blue stained bands correspondingto full-length PapD which are shown.

B: PapD incubated with the G1′-19′WT, G1′-16′WT, K1′-19′WT, andG2′-21′amide was added to reduced, denatured PapD-PapG and the amount ofPapD-PapG complex restored in each sample was quantitated as describedin example 2. The % inhibition represents the amount of the PapD-PapGcomplex restored with peptide-treated PapD compared to the amount ofPapD-PapG complex restored with untreated PapD. The graph represent theaverage of 4 experiments. PapD and PapD-PapG complex were prepared asdescribed in F. Lindberg et al, J. Bacteriol. 171, 6052 (1989) and S. J.Hultgren et al., Proc. Natl. Acad. Sci. USA 86, 4357 (1989).

FIG. 3 Stereoscopic view of the three-dimensional structure of PapDco-crystallized with the G-19WT peptide determined to 3.0 Å resolution.The peptide is bound in an extended conformation along the G1 β strandin the cleft of PapD with the terminal carboxylate group forminghydrogen bonds with residues Arg-8 and Lys-112 of PapD.

FIG. 4 Stereoscopic view of the 3.0 Å resolution electron density of theG1-19WT peptide COOH-terminus and neighbouring PapD residues,superimposed on the refined structure. The electron density map wascalculated with coefficients (2|Fo|−|Fc|) and contoured at 1σ.

FIG. 5 Stereoscopic view of a PapD-peptide complex showing itsinteraction with a twofold non-crystallographically related complex.

FIG. 6 Superposition of COOH-terminal domains of native PapD (thicklines) and complexed PapD (thin lines) showing the 13° difference in thehinge bend angle between the two structures. The structures weresuperimposed using the LSQ option in the graphics program O. Theresulting rms for 98 Ca atoms of the COOH terminal domain was 0.66 Å.

FIG. 7: Ability of PapD Arg-8, Lys-112 and Thr-7 mutants to bind PapGand restore the PapD-PapG complex in vitro. 0.4 μg of PapD-PapG complexwas reduced and denatured as described in FIG. 2B. The denaturedPapD-PapG complex was then diluted with 0-630 ng purified wild type (WT)or mutant PapD. The amount of PapD-PapG restored in the samples wasdetermined as in FIG. 2B and was graphed as a percentage of the greatestamount of PapD-PapG restored. The values graphed represent the averageof 3 experiments. Wild type and mutant PapD and the PapD-PapG complexwere purified as described in F. Lindberg et al, J. Bacteriol. 171, 6052(1989) and S. J. Hultgren et al., Proc. Natl. Acad. Sci. USA 86, 4357(1989).

FIG. 8: 6-hydroxydopamine, hdo_(—)0, and other members of the hdofamily. hdo_(—)0 Was used as starting-point for the construction of afamily of compounds intended to bind to the binding-site of PapD.hdo_(—)4: “Aryl” denotes 3,4-dihydroxyphenyl

hdo_(—)6: “Aryl” is a 3,4-dihydroxyphenyl group.

hdo_(—)7: “Aryl” is a 3-hydroxyphenyl group.

hdo_(—)8: “Aryl” is a 3-nitrophenyl group.

hdo_(—)9: “Aryl” is a phenyl group.

FIG. 9: The hdo family and its interaction with PapD.

A: “Aryl” is a phenyl group, a heteroaromatic ring, or a substitutedphenyl group with polar substituents unsymmetrically in order to obtainhydrophobic contact with the protein on one side and interaction withsolvent water on the other.

B: Examples of “Aryl” wherein R is hydrogen or alkyl; “X” is an oxygenatom, a sulphur atom, or a amino group; and CG″ is a negatively chargedgroup, such as a carboxyl group, a tetrazoyl group, a phosphate group, aphosphate ester or a sulphate group.

C: Examples of the group CG.

FIG. 10: General reaction scheme for the production of various membersof the hdo family.

FIG. 11: General reaction scheme for the production of various membersof the bpy family.

FIG. 12: General reaction scheme for the production of various membersof the bpy family.

FIG. 13: General reaction scheme for the production of various membersof the bpy family.

FIG. 14: General reaction scheme for the production of various membersof the bpy family.

FIG. 15: General reaction scheme for the production of various membersof the bpy family.

FIG. 16: General reaction scheme for the production of various membersof the bpy family.

FIG. 17: Graph over the inhibitory effect of different lengths ofC-terminal peptide fragments of PapG on the binding between PapD and thefusion protein MBP-G1′-140′.

The G1′-8′ fragment exhibits a significantly higher inhibitive effect onthe binding than does the shorter G1′-6′ and G1′-7′ fragments.

FIG. 18: Graph over the inhibitory effect of different analogues ofG1′-8′ (wherein the 8 amino acid residues one at a time has beenreplaced by serine (S) or Alanine (A)) on the binding between PapD andthe fusion protein MBP-G1′-140′.

The effect of the replacements of residues 4′, 5′ and 6′ in G1′-8′reveals that these residues are important in the interaction with PapD,as the replacement of these residues results in less inhibitorypeptides.

FIG. 19: Graph over the inhibitory effect of different analogues ofG1′-8′ (wherein the 8 amino acid residues one at a time has been deletedsimultaneously with the addition of an N-terminal serine) on the bindingbetween PapD and the fusion protein MBP-G1′-140′.

Also in this experiment, the significance of amino acid residues 4′, 5′,and 6′ are emphasized, as the deletions at these positions leads to lessinhibitory peptides.

FIG. 20: MBP/G fusion constructs, PapG-truncates and synthetic peptidesused in example 10.

The open box indicates the primary sequence of PapG. The positions ofthe four Cys residues are shown. The hatched bar represents MBP. Thestarting and ending residues of PapG fused onto the COOH-terminus of MBPare noted for each fusion. The terminating residue for eachPapG-truncate is also indicated. The names of the MBP/G fusion proteinsare listed. In the lower portion of the figure, the solid boxes localizethe PapD interactive sites and the sequences of the four peptides usedin the example are listed (cf. also SEQ ID NOS: 19-22).

FIG. 21: PapD-MBP/G interactions in vivo.

Periplasmic extracts containing PapD and each MBP/G fusion weresubjected to amylose affinity chromatography. The eluates were analyzedon A) a 12.5% Coomassie blue stained SDS polyacrylamide gel; B) awestern blot using anti-PapD antiserum; or C) a silver stained IEF gel.In FIGS. 21A, B and C, samples were purified form periplasmic extractscontaining PapD and MBP (lane 1), PapD and MBP-G1′-19′ (lane 2), PapDand MBP-G1′-81′ (lane 3), PapD and MBP-G1′-140′ (lane 4). The positionof co-purified PapD is indicated. MBP alone and MBP/G fusion truncatesalso co-purified with the MBP/G fusions. The molecular weight of theslowest migrating bands on SDS-PAGE correspond to each full length MEP/Gfusion protein. On the IE F gel (C) several bands could be seen for thesame reasons. A unique band at pI 5.2 was detected in FIG. 2C, lane 4.This band was excised, boiled in SDS sample buffer and analyzed bywestern blotting with anti-PapD and anti-MBP antisera. It was composedof both MBP-G1′-140′ and PapD (FIG. 2D, lane 1).

FIG. 22: Inhibition of chaperone function by expression of MBP/G fusionsin vivo.

Strains carrying pFJ22 (papDJKEFGA) and plasmids encoding the MBP/Gfusions were induced with 1 mM IPTG. Periplasmic extracts containingpilus subunits and MBP (lane 1), or MBP-G1′-19′ (lane 2), or MBP-G1′-81′(lane 3), or MBP-G1′-140′ (lane 4) were analyzed by western blottingusing anti-PapA antiserum (A), or anti-PapD-PapG antiserum (B), oranti-tip fibrillum antiserum (C). In this assay the presence of thesubunit indicates that chaperone-subunit interactions occurred sincesubunits are degraded in the absence of an interaction with thechaperone. Note that the presence of the PapA, PapG and PapF subunitsdecrease significantly when co-expressed with MPB-G1′-140′.

FIG. 23: Binding of PapD to MBP/G fusion proteins in vitro.

(A) PapD was incubated with 1 μg of amylose affinity purified MBP(lane), MBP-G1′-19′ (lane 2), MBP-G1′-81′ (lane 3), or MBP-G1′-140′(lane 4) and complex formation was eveluated on the silver stained IEFgel. Positions of MBP/G fusions, PapD and the PapD-MBP-G1′-140′ complexare indicated.

(B) Amylose affinity purified MBP/G fusion proteins were coated to wellsof microtiter plates. The concentration of the MBP/G proteins in thewells is indicated. Binding of 50 pmol/50 ml of PapD to the immobilizedproteins was determined by ELISA using anti-PapD antiserum.

FIG. 24: Identification of PapD-PapG2 truncate complex by acidic nativegel electrophoresis.

Periplasm containing PapD and PapG, PapG2 or PapG3 (lane 2, 3 and 4respectively) were subjected to Gal α(1-4) Gal chromatography and etheluates were analyzed on acidic native gel electrophoresis followingwestern blotting using anti-PapD (A) and anti-PapG (B) antisera.Purified PapD-PapG complex was loaded in lane 1.

FIG. 25: Characterization of a second site on PapG recognized by PapD.

Four synthetic peptides overlapping the PapG region from residue 156′ to120′ (indicated in FIG. 20) were coated to wells of microtiter plates.The concentration of each peptide in the wells is indicated. Binding of200 pmol/50 ml of PapD to the immobilized peptide was determined byELISA using anti-PapD antiserum.

EXAMPLES Examples 1

Identification of the Motif of Binding Between PapD and G1′-19 ′WT

Materials and Methods

PapD was prepared as described by A. Holmgren et al. in J. Mol. Biol.203, 279 (1988) and obtained from the Department of MolecularMicrobiology, Washington University School of Medicine, St. Louis, USA.

The peptide G1′-19′WT was prepared using Fmoc solid phase synthesisfollowed by purification by reversed phase HPLC. The peptide wasobtained form Department of Chemistry, University of Lund, Lund, Sweden.Table 1 indicates the peptides used in binding assays in thisapplication.

In the present application, amino acid residues or peptide fragmentsoriginating from other peptides than PapD (e.g. from PapG or from PapK)will be indicated with ′, e.g., “G1′-19′WT” or “Pro-1′”, in order todistinguish such residues from amino acid residues of PapD. Further, thenumbering is from the C-terminal end of the non-PapD peptide, i.e.G1′-19′ denotes the 19 C-terminal amino acid residues of PapG.

Cxystallisation of PapD-peptide Complex

Crystals of the PapD-peptide complex were obtained by vapour diffusionagainst 20% PEG8000, 0.1 M cacodylate buffer at pH 5.0, and 0.2 Mcalcium acetate. The crystallisation drop contained equal volumes ofreservoir and protein solutions. The protein solution (17 mg/ml)contained a 1:1 molar ratio of PapD and peptide in 20 mM MES(2-[N-Morpholino]ethane sulphonic acid) at pH 6.5 with 1.0% β-octylglucoside.

X-ray Crystallography

The obtained crystals of the complex between PapD and the protein weremounted inside sealed quartz-glass capillary tubes and initiallycharacterised by examining them on a X-ray precession camera therebyobtaining pictures of the X-ray diffraction pattern on photographic filmrepresenting spatially non-distorted images of the reciprocal lattice ofthe crystals. Using standard analysis of the images, it was determinedthat the crystalshave a monoclinic space group, C2, with celldimensions: a=130.7 Å, b=83.5 Å, c=59.2 Å, and β=117.2°, two moleculesin the asymmetric unit and diffract to 2.9 Å resolution on a lab X-raysource with a rotating anode and Cu K_(α) target.

Collection and Processing of Experimental Data

In order to solve the atomic structure of molecules by X-raycrystallography, the positions and intensities of the diffraction maximahave to be measured. Intensity data for the PapD-peptide crystals werecollected on a Huong-Hamlin multiwire area-detector system (Xuong etal., 1985). All data were obtained from a single crystal and initiallyprocessing was carried out using the MADNES software package(Messerschmit and Pflugrath, 1987). Merging and scaling of the data wascarried out using ROTAVATA and AGROVATA from the CCP4 package (see CCP4,The SERC (UK) Collaborative Project No. 4, A Suite of Programs forProtein Crystallography, Darebury Laboratory, UK). The final data setcontained 9592 independent reflections with an Rsym of 6.8% for databetween 20.0 and 3.0 Å resolution$\left( {{{Rsym} = {\sum{\sum\frac{\left( {{I_{hi}} - {I_{h}}} \right)}{\sum\limits_{hi}I_{hi}}}}},} \right.$

where I_(hi) and I_(h) are the intensities of the individual and meanstructure factors, respectively).

Solution of Three-dimensional Structure

The structure of the PapD-peptide complex was solved using the programXPLOR performing the standard method of molecular replacement. Thesearch model used was the refined 2.0 Å resolution structure of PapD.Using 8.0 Å to 4.0 Å resolution data, the self-rotation function gave aclear non-crystallographic two-fold axis, and the top two peaks of thecross-rotation gave the correct solution which was improved usingPatterson correlation (PC) refinement. The correct solution was alsoobtained from the top peaks in the translation functions, the R-factorbeing 39.0% for 8.0 to 4.0 Å resolution data (the R-factor defined as$\left. {{R - {factor}} = \frac{{\sum{{Fo}}} - {{Fc}}}{\sum{Fo}}} \right).$

Refinement was obtained with subsequent rigid body refinement in whichall four domains of the 2 PapD molecules in the asymmetric unit wereallowed to refine independently resulting in a R-factor of 36.4% for thesame data.

Examination of an |Fo|−|Fc| electron density map at this stage using thegraphics program O showed clear density corresponding to the peptide inthe PapD cleft and running along the surface of the protein. Theorientation of the peptide was easily determined from the electrondensity, but initially only the final 10 C-terminal amino-acids of thepeptide could be into density.

Refinement and Analysis of Structure

Simulated annealing refinement with XPLOR was initiated at this stage.Several additional cycles of model building and refinement were carriedout with a further 4 peptide amino-acids being added to yield anR-factor for the current model of 18.2% for 8.0 to 3.0 Å resolutiondata. The model at the present stage of refinement (which contains nowater molecules and does not include the first 5 N-terminal amino-acidsof the peptide) has root-mean square (rms) deviations from idealgeometry of 0.020 for bonds lengths and 4.2° for bond angles.

The peptide is seen to bind in an extended conformation with theC-terminal pro-1′ anchored within the inter-domain cleft and proposed(6) subunit binding site. Hydrogen bonds are formed between the peptidecarboxy terminus and two invariant positively charged residues of PapD,Arg-8 and Lys-112. Site-directed mutagenesis has now confirmed thatArg-8 and Lys-112 are essential for the binding of pilin subunits bothin vitro and in vivo (cf. example 2)). The Pro-1′ side chain makes vander Waals contacts in the cleft with residues form both domains of PapD:Thr-7, Thr-152, Ile-154, Thr-170and Ile-194. The neighbouring peptideresidue, Phe-2′, lies in a shallow pocket formed between the twoβ-sheets of the N-terminal domain and makes hydrophobic interactionswith Leu-4, Thr-7, Thr-109 and Ile-111. The peptide then runs along thesurface of the N-terminal domain, forming a parallel β-strandinteraction with strand G1. In this way 7 main chain hydrogen bonds areformed between Met-8′ to Phe-2′ of the peptide and Gln-104 to Lys-110 ofPapD, thus extending the β-sheet of PapD out into the peptide.

Apart form the C-terminal residues Phe-2′ and Pro-1′ there arerelatively few contacts between the side chains of the peptide and PapD.The major interactions are provided by the main chain hydrogen bonds tostrand G1. There are, however, a number of hydrophobic interactionswithin the β-sheet, in particular between the peptide's Met-8′ andMet-6′ with Leu-103, Ile-105 and Leu-107 of strand G1.

Calculations revealed that the four hydrophobic peptide side chains ofresidues 2′, 4′, 6′, and 8′ contribute 20% of the total buried surfacearea (582 Å²) between the peptide and the protein. Therefore, eventhough the major stabilization of the complex is provided via hydrogenbonding, hydrophobic interactions are not insignificant, and it isbelieved that they provide part of the explanation for the specificityof PapD for pilus related peptides and subunits. Experimental support ofthis theory is provided by the reduced binding of PapD to the peptideG2′-21′amide as compared to the G1′-19′WT peptide (FIG. 1B and table C).Hydrogen bonding of the COOH-terminus of the G2′-21′amide (which lacksPro-1′) to Arg-8 and Lys-112 of PapD allows main chain hydrogen bonding,but dislocates the four hydrophobic side chains in the peptide fromtheir subsites in PapD, resulting in a reduction in binding strength.

Within the crystal the PapD-peptide β sheet was extended even further asa result of non-crystallographic twofold symmetry which placed a secondPapD-peptide complex adjacent to the first so that the two bound peptidechains interacted as antiparallel β strands. In the present model, eightmain chain hydrogen bonds are formed between the two peptides; see thefollowing table:

TABLE B PapD₁ Peptide₁ Peptide₂ PapD₂ Pro-1′-COOH Arg-8-NH₂ Pro-1′-COOHLys-112-NZ Phe-2′-NH Lys-110-C═O Glu-13′-NH Ser-3′-C═O Glu-13′-C═OSer-3′-NH Leu-4′-C═O Lys-110-NH Leu-4′-NH Gln-108-C═O Ser-11′-NHVal-5′-C═O Ser-11′-C═O Val-5′-NH Met-6′-C═O Gln-108-NH Met-6′-NHAla-106-C═O Ser-9′-NH Thr-7′-C═O Ser-9′-C═O Thr-7′-NH Gln-104-C═OMet-8′-NH Met-8′-C═O Ala-106-NH Ala-106-NH Met-8′-C═O Met-8′-NHGln-104-C═O Thr-7′-NH Ser-9′-C═O Thr-7′-C═O Ser-9′-NH Ala-106-C═OMet-6′-NH Gln-108-NH Met-6′-C═O Val-5′-NH Ser-11′-C═O Val-5′-C═OSer-11′-NH Gln-108-C═O Leu-4′-NH Lys-110-NH Leu-4′-C═O Ser-3′-NHGlu-13′-C═O Ser-3′-C═O Glu-13′-NH Lys-110-C═O Phe-2′-NH Lys-112-NZPro-1′-COOH Arg-8-NH₂ Pro-1′-COOH

A mixed β sheet is thus created between the two complexes and extendsover ten β strands (FIG. 5). No contacts are observed between the twonon-crystallographically related PapD molecules themselves, both ofwhich were positioned in similar environments within the crystal andpossessed a similar number of intermolecular contacts. The calculatedburied surface area between the two non-crystallographically relatedpeptides was 520 Å², a value similar to the surface area buried betweenthe protein and peptide. This “dimerization” appears to be a consequenceof crystal packing, because all evidence shows that PapD forms monomericcomplexes with peptides or with intact PapG in solution.

The hydrogen bonding pattern between PapD and the peptide breaks atSer-9′, but the peptide remains in van der Waals contact with PapD untilSer-11′ where the peptide runs beyond the F1-G1 loop, but remainshydrogen bonded to the non-crystallographically relatedpeptide as far asGlu-13′ (table B). The last resolved amino acid of the peptide isGly-14′ which is positioned close to the binding cleft of thenon-crystallographically related PapD. the first five amino terminalamino acids, including three positively charged residues, had no densityand therefore must have been disordered in the crystal structure. Inagreement with this, the peptide's amino terminus is not important forbinding to PapD in solution, since the G1′-7′WT peptide lacking theamino terminal 12 amino acids is found to be an effective inhibitor ofPapD binding to the immobilized G1′-19′WT peptide (table C and example2).

The structures of the individual PapD domains in the peptide complex areessentially the same as those of native PapD (Holmgren and Branden,1989). However, there is a significant movement of the domains withrespect to each other with a 13° jaw-closing or hinge bending motionmaking the angle of the PapD boomerang more acute (FIG. 6). Whether ornot this conformational change is the result of binding of peptide ordifferent crystal packing between the two crystal structures is unclear.

In the native PapD structure the electron density obtained for the longF1-G1 loop is poor between residues 96 and 102, suggesting that it israther flexible and disordered in the crystal. In the peptide complex,however, this loop is better resolved, indication that binding of thepeptide makes this loop more rigid. Superimposing the NH₂-terminaldomains of native PapD and the peptide complex shows that there is alsoa significant difference in the F1-G1 loop position between the twostructures (rms for the 110 NH₂-terminal Cα atoms is 1.84 Å, with amaximum main chain movement of about 9 Å for Leu-103). In the peptidecomplex the loop is seen to twist at one end away form the β barrel ofthe NH₂-terminal domain thus facilitating a more extensive contactbetween strand G1 and the peptide. As with the hinge-bending of the twodomains, it is not yet possible to say with certainty whether this loopshift is a consequence of peptide binding or of crystal packing; therather open conformation of the F1-G1 loop suggests that it may belargely the latter. Nevertheless, evidence that similar interactionbetween PapD and peptides or pilus subunits occur in solution isprovided by protease protection experiments after the F1-G1 loop of PapDis protected from tryptic cleavage by the binding of both native PapGand the G1′-19′WT peptide (example 2 and FIG. 2A).

Example 2

Binding Between PapD and the Carboxyl Terminus of Other Pap Peptides

G1′-19′WT, E1′-19′WT, F1′-19′WT, K1′-19′WT and H1′-19′WT peptides (seetable 1) were synthesized corresponding to the 19 carboxyl terminalresidues of P pilus subunit proteins PapG, PapE, PapF, PapK and PapH,respectively (see: Grant et al., 1992, and references cited therein).

TABLE 1 Name SEQ ID NO: Peptide sequence G1′-19′WT 6   NH₂-GKRKPGELSGSMTMVLSFP-COOH G1′-16′WT 7      NH₂-KPGELSGSMTMVLSFP-COOH G1′-11′WT* 8           NH₂-SGSMTMVLSFP-COOH G1′-7′WT 9               NH₂-TMVLSFP-COOH G1′-19′SV 10   NH₂-GKRKPVELSGSMTMVLSSP-COOH G1′-19′amide 11  NH₂-GKRKPGELSGSIMVLSFP-CONH ₂ G2′-21′amide 12NH₂-EEGKRKPGELSGSMTMLSF-CONH ₂ E1′-19′WT* 13   NH₂-QNLIAGPFSATATLVASYS-COOH H1′-19′WT 14   NH₂-KKLEAGNYFAVLGFRVDYE-COOH K1′-19′WT 15   NH₂-KSVVPGDYEATATFELTYR-COOH F1′-19′WT 16   NH₂-GILNGGDFQTTASMAMIYN-COOH MS 17       NH₂-YALAPNAVIPTSLALL-COOH*Water insoluble peptides

The residues in the peptides were numbered starting with the carboxylterminal residue as 1, and ending with the amino terminal residue.Peptides were also synthesized which deviated in length (G1′-16′WT,G1′-11′WT, G1′-7′WT) or sequence (G2′-21′amide, G1′-19′SV, G1′-19′NH₂)from the wild type PapG carboxyl terminal sequence. Using anenzyme-linked immunosorbent assay (ELISA), we tested the ability of PapDto bind to each peptide coated on wells of microtiter plates:

5 mg/ml stock solutions of peptides in water or 50′ acetic acid werediluted to a concentration of 2.5 pmol/50 μl in PBS. 50 μl of thepeptide solution was coated overnight onto microtiter wells (Nunc-IrmunoPlate Maxisorp) at 4° C. The solutions in the plates were discarded, andthe wells were blocked with 200 μl 3% bovine serum albumin (BSA, Sigma)in PBS (120 mM NaCl/2.7 mM KCl/10 mM phosphate buffer salts, pH 7.4) for2 hours at 25° C. The plates were washed vigorously 3 times with PBS andincubated with 50 μl of the indicated amount of purified PapD (Lindberget al, 1989). After 3 washes with PBS, the wells were incubated with a1:500 dilution of anti-PapD rabbit antiserum (Lindberg et al, 1989) in3% BSA/PBS for 45 min. at 25° C. After 3 washes with PBS, the wells wereincubated with a 1:1000 dilution of goat anti-rabbit IgG coupled toalkaline phosphatase (Sigma) in 3% BSA/PBS for 45 min. at 25° C.Following 3 washes with PBS and 3 washes with developing buffer (10 mMdiethanolamine pH 9.5, 0.5 mM MgCl₂), 50 μl filtered p-nitrophenylphosphate substrate (Sigma) in developing buffer (10 mg/ml) was added.The absorbance at 405 nm was read after 60 min of incubation in the darkat 25° C.

In addition, the ability of water-soluble peptides to inhibit PapDbinding to G1′-19′WT-coated wells in a soluble inhibition ELISA weretested, since peptide conformations may have been affected by binding tothe plastic microtiter plates:

Microtiter wells were coated overnight at 4° C. with 50 μl of 2,5pmol/50 μl of the G1′-19′WT peptide. The wells were washed with PBS andblocked with 3% bovine serum albumin (BSA). A 25-fold molar excess ofeach test peptide was pre-incubated with 100 pmoles PapD for 30 min. andthe PapD-peptide solution was then added to the coated wells andincubated at 25° C. for 45 min in the presence of 3% BSA/PBS. Thesubsequent primary antibody, secondary antibody and developing steps aredescribed above. The ability of the peptides to inhibit binding of PapDto the G1′-19′WT peptide was calculated by dividing the amount of PapDbinding in the presence of peptide with the amount of PapD binding inthe presence of water. 0% inhibition includes values where binding wasgreater than that of PapD pre-incubated with H₂O.

The results of PapD binding to wild type, variant and different lengthpeptides are shown in FIGS. 1A, B and C.

As can be seen the peptides bound well to the PapG peptide, moderatelyto PapE, PapF and PapK, whereas there was no binding to PapH and therandom hydrophobic peptide, MS. (FIG. 1A). These results suggest thatthe chaperone recognizes PapG, PapE, PapF and PapK in part by binding tothe carboxyl terminus of these subunits. The inability of PapD tointeract with the PapH peptide indicates that PapD binds differently toPapH, possibly due to PapH's function as a polymerization terminator(Baga et al., 1987). The ability of a peptide to inhibit PapD binding toimmobilized G1′-19′WT generally corresponded to the affinity that PapDhad for the respective immobilized peptide (FIG. 1, % inhibition),arguing that the interaction of PapD with soluble and immobilizedpeptides is similar.

As can be seen from example 1, the molecular basis of the PapD-peptideinteractions have been studied by co-crystallization of PapD with theG1′-19′WT peptide. In summary, the 19 amino acid peptide is anchored inthe chaperone cleft by hydrogen bonds between the peptide's carboxyterminus and Arg-8 and Lys-112 which are invariant in all periplasmicchaperones (Holmgren et al., 1992). The peptide bound to the G1 strandof PapD as a parallel β-strand, forming at least 10 backbone hydrogenbonds, and resulted in an ordering of the F1 to G1 loop.

Replacement of the carboxyl terminal proline on the PapG peptide with anamide (G2′-21′amide) abolished binding to PapD in solution and reducedbinding of PapD to the immobilized peptide by approximately 759. (FIG.1B). In contrast, substituting only the carboxylate group with an amideto create the G1′-19′NH₂ peptide did not affect binding to PapD ineither the immobilized or soluble inhibition ELISA assays (FIG. 1B).These results indicate that the terminal proline probably is required toposition the carboxylate group so that it can form hydrogen bonds withthe invariant Arg-8 and Lys-112 cleft residues.

PapD also binds to the immobilized shorter peptides G1′-16′WT andG1′-11′WT and the G1′-16′WT and G1′-7′WT peptides inhibited binding ofPapD to the immobilized G1′-19′WT peptide in solution. Table C shows theability of a 25-fold molar excess of the water soluble peptides toinhibit binding of 100 pmol/well of PapD to G1′-19′WT-coated wells. The% inhibition represents the percentage of PapD binding in the presenceof the peptide compared to in the presence of water and are the averageof two experiments performed in duplicate:

TABLE C Peptide % Inhibition G1′-19′ 63 G1′-7′ 49 G1′-4′ 0 MS 1G2′-21′amide 0 G1′-19′amide 56

PapD does not bind to immobilized G1′-7′WT peptide, probably becausethis peptide is to short to bind to the microtiter well as well as PapD.It, thus seems that as few as 7 carboxyl terminal residues are necessaryfor PapD binding of a peptide. Together these results support a modelwhere, in addition to the anchoring interaction of the carboxylate groupin the PapD cleft, seven carboxyl terminal residues are required for“zippering” the peptide along the G1 strand of PapD during PapD-peptidecomplex formation.

Conserved residues phenylalanine and glycine residues at positions 2 and14 from the carboxyl terminus were substituted by serine and valine,respectively, to create the peptide G1′-19′SV and decreased binding toPapD by 36%. This could result form less efficient coating orpresentation in the microtiter plate since G1′-19′SV was as efficient asoluble PapD inhibitor as the peptide G1′-19′WT (FIG. 1B). Since theseresidues are critical for incorporation of PapG into the pilus, it isbelieved that they be more important for intersubunit polymerizationinteractions and pilus assembly than in PapD subunit interactions invivo.

Partial digestion with trypsin cleave PapD in the F1-G1 loop at residuesLeu-103 and Lys-99, respectively:

400 μg of PapD was partially digested by incubation with either 4.5 μgtrypsin or 0.45 μg chymotrypsin on PBS for 20 min. at 37° C. The PapDdigests were applied to a C-18 HPLC column (Beckman) and 2 majorfragments were eluted with a 0-100% acetonitrile gradient in 0.01%trifluoroacetic acid. The PapD fragments were identified by theirmolecular weight on SDS-PAGE and amino-terminal sequencing. TheN-terminal amino acid sequences of the approximately 14 kDa tryptic andchymotryptic fragments were identified as residues 100-108 and 104-109of PapD, respectively, corresponding to cleavage after Lys-99 (fortrypsin) and Leu-103 (for chymotrypsin). The N-terminal sequences of the11 and 12 kDa bands in the digests were identical to the N-terminalsequence of PapD.

The G1′-19′WT, G1′-16′WT and K1′-19′WT peptides, but not theG2′-21′amide peptide, reduced the rate of tryptic cleavage of PapD overtime (FIG. 2A). These data argue that binding of PapD to the peptidealtered contacting the loop and thereby protectin to form cleavage. Thiseffect may be related to the ordering of the F1-G1 loop of PapD observedin the PapD-peptide crystal structure, and suggests that, in solution,both peptides extends along the G1 β-strand and interact with the F1-G1loop of PapD.

As described in Kuehn et al., Proc. Natl. Acad. Sci. USA 88, 10586(1991), native PapD is able to bind to reduced, denatured PapG andrestore the PapD-PapG complex in vitro. This reconstitution assay wasused to determine the ability of the peptides to inhibit PapD activityin vitro. The limited solubility of the G1′-7′WT peptide unfortunatelyhas prevented the testing of this peptide in the assay. Increasingamounts of the G1′-19′WT, G1′-16′WT and K1′-19′WT peptides inhibitrestoration of the PapD-PapG complex by PapD, while the G2′-21′amidepeptide has no effect (FIG. 2B). The ability of the G1′-19′WT, G1′-16′WTand K1′-19′WT peptides to prevent PapD form binding to PapG indicatedthat these peptides bound in the subunit binding site of PapD.

The ability of peptides to inhibit restoration of the PapD-PapG complexby PapD was tested as follows.: 0.3 μg of PapD-PapG complex was reducedand denatured by incubation at 25° C. for 20 min with 4 M urea/10 mMdithiothreitol (DTT), 1.2 μg (50 pmol) of PapD was incubated at 25° C.for 10 min with 5-14.5 μg (2.5-7.25 nmol) of peptide. The PapD-peptidesolution was then added to the reduced, denatured PapD-PapG, incubatedat 25° C. for 10 min, and applied to an IEF 3-9 gel (Pharmacia). Theamount of PapD-PapG restored in each sample was quantitated bydensitometry of the silver stained IEF band corresponding to the pI ofthe DG complex.

Site directed mutations in strictly conserved cleft residues of PapDpredicted to be critical in the PapD-peptide interaction wereconstructed to test whether the PapD-peptide crystal structure is areflection of part of the PapD-pilus subunit interaction interface (thepositions of the mutations are indicated in FIG. 3). Highly conservedThr-7 was changed to a valine (Thr-7-Val) in order to test whether itshydroxyl group formed hydrogen bonds critical for PapD binding tosubunits.

This mutation removed the hydroxyl group while maintaining the stericvolume of the side chain. Mutations in Lys-112 and

Arg-8 were designed to test whether hydrogen bonding to the terminalcarboxylate group of pilus subunits is a critical feature of thechaperone recognition process. The invariant Lys-112 residue was changedto an alanine (Lys-112-Ala) to remove the charged side chain and to amethionine (Lys-112-Met) to replace the charged group with ahydrophillic group while maintaining the side chain packing. Theinvariant Arg-8 has been shown previously to be required tor the abilityof PapD to bind subunits and mediate pilus assembly in vivo (Slonim etal, 1992). Glu-167/E167, a variable residue in domain 2 of PapD (FIG.3), does not appear to be involved in the interaction between PapD andthe peptide in the crystal structure and mutations in this residue havebeen shown to have little or no effect on PapD function in vivo (Slonimet al, 1992). E167 was changed to histidine (E167H) to test whether thisnegatively charged residue at the lip of domain 2 has any role in thePapD-peptide interaction. All of the mutants were secreted into theperiplasmic space as stable proteins similar to wild type PapD. Inaddition, the elution profile form a cation-exchange FPLC column and theelectro-phoretic properties of the purified mutant PapDs were similar tothe wild type protein, supporting the prediction that these mutationswould not affect the overall structure of PapD (data not shown).

The ability of the mutant chaperones to bind pilus subunits and modulatepilus assembly in vivo was correlated to their ability to bind to theG1′-19′WT peptide and to PapG in vitro. Wild type PapD bound theG1′-19′WT peptide to cause a mobility shift towards the negativeelectrode in a native polyacrylamide gel assay (Lam and Calderwood,1992), probably due to a net positive charge increase in thePapD-peptide complex. In contrast, when Arg-8-Gly, Arg-8-Ala andLys-112-Ala PapD mutants were incubated with the G1′-19′WT peptide theydid not cause a mobility shift in the native-PAGE assay, indicating thatthese mutations in PapD abolished peptide binding. Similarly, mutationsin Arg-8 and Lys-112 abolished the ability of PapD to bind to PapG andreconstitute the

PapD-PapG complex in vitro (FIG. 7) and to bind to pilus subunits andmediate pilus assembly in vivo (cf. table 2).

TABLE 2 Residue HA Subunit stabilization^(#) class Mutation titer* Pilusassembly⁺ PapA PapE PapG PapF PapK Invariant Arg-8-Gly None − − − − − −Arg-8-Ala None − + − + − + Arg-8-Met None − ++ − ++ − ++ Lys-112-AlaNone − + − + − + Lys-112-Met None − ++ − − − − Conserved Thr-7-Val 64+++ ++++ ++++ ++++ ++++ ++++ Variable Glu-167-X^(§) 128 ++++ ++++ ND ND++++ ++++ *The hemagglutionation (HA) titer was quantitated afterinduction with 0.01 mM IPTG. The HA titer represents the greatestbacterial dilution which still agglutinates erythrocytes. HB101/pPAP37expressing wild type PapD has an HA titer of 128. ⁺The amount of piliassembled were released from the cells and quantitated after inductionwith 0.01 mM IPTG. “−” indicates no pili were detected; the degree ofpiliation of HB101/pPAP37 expressing wild type PapD corresponds to“++++”. ^(#)Chaperone-assisted periplasmic stabilization of pilussubunits PapA, PapE, PapF, PapK and PapG was determined as describedpreviously (Slonim et al., 1992); “−” indicates no stabilization of thesubunit; the number of +'s indicate the degree of subunit stabilizationas compared to wild type PapD. ND indicates not determined. ^(§)Xindicates mutations to His, Asp, Thr, or Gly.

The remarkable consistency of the in vitro and in vivo results and thecrystal structure argue that anchoring of pilus subunits by conservedresidues Arg-8 and Lys-112 is a critical part of the interaction betweenPapD-like chaperones and pilus biogenesis.

Site-directed mutagenesis was performed using the Bio-Rad in vitromutagenesis kit following the manufacturers instructions. The followingprimers (non-coding strand) were used to introduce mutations in the papDgene (SEQ ID NO: 1):

Thr-7-Val 5′-GTCAAACACCGCCGGAACTCGTCCAGGCGA-3′  SEQ ID NO: 3

 Lys-112-Ala 5′-CGGGCGATAAAAAAGAGCTATTTTGGTCTG-3′  SEQ ID NO: 4

Lys-112-Met 5′-GCGATAAAAAAGCATTATTTTCCTCTG-3′  SEQ ID NO: 5

Mutations in papD were confirmed by sequencing and the altered papDgenes were cloned into vector pMMB91 under the inducible P_(tac)promotor as previously described for other papD mutations (Slonim et al,1992). The plasmids were named pThr-7-Val, pLys-112-Ala and pLys-112-Metaccording to the residue number and mutated amino acid. Plasmid pLS101is an isogenic construct containing the wild type papD gene; plasmidpE167H, pR8G and pR8A contain genes encoding PapD with point mutationschanging Glu 167 to His and Arg-8 to Gly and Ala, respectively.

The data presented in this example and example 1 demonstrate that thePapD-peptide interactions found in the crystal structure reflects thePapD-pilus subunit interaction interface in vivo. These interactionsdefine for the first time a mechanism whereby chaperones utilize theirimmunoglobulin-like domains in a novel recognition paradigm to bind to adiverse group of proteins. PapD residues Arg-8 and Lys-112, located inβ-strands in the cleft between the two domains, play crucial roles inchaperone recognition by servings as molecular anchors for the carboxylterminus of pilus proteins. The fact that Arg-8 and Lys-112 are highlyconserved amongst periplasmic chaperones indicates that they probablyhave a universal role in forming the “active site” that binds to theterminal carboxylate group of other protein subunits. Backbone hydrogenbonds along the β-strand of PapD subsequently provide strong,sequence-independent interactions along the length of the carboxylterminus of the subunit. The “zippering” interaction of the conservedalternating hydrophobic residues of the carboxyl terminus of the pilussubunit with the conserved alternating hydrophobic residues in the G1β-strand of the chaperone adds strength and specificity to the binding.Therefore, unlike other immunoglobulin-like proteins (Amit et al, 1988and de Vos er al, 1992), PapD utilizes the β-strand and interdomaincleft features of its immunoglobulin-like structure to provide arecognition mechanism for binding to several different proteins.Variable residues in loop regions of antibodies provide the exquisitespecificity necessary from an extensive antibody binding repertoire.Similarly, residues in the F1-G1 loop of PapD may provide specificity tochaperone binding since these have been found to vary in length andcomposition amongst members of the periplasmic chaperone family(Holmgren et al, 1992).

Example 3

Designing of Compounds Capable of Binding to the Binding Site of PapD

Having determined the location of a promising binding site forinhibitory ligands as described in Examples 1 and 2, the computerprograms ‘PLIM’ and ‘PLIM_DBS’ (developed by Symbicom AB) were used tofind templates for families of compounds capable of binding to thebinding site (see the general description above).

PLIM was run on a 20 Å box around the Arg-8 region of thehigh-resolution structure of PapD, searching for low-energy bindingpositions for NH, NH₂, O, OH and C probes. The PLIM runs resulted in anumber of suggested positions and orientations of favourable chemicalgroups (site points) in the region near Arg-8 and Lys-112.

Approximately 50 sites were identified, and then triplets and quartetsof these site points were selected for their mutual compatibility, thatis to say they must have appropriate geometric relationships—distancesand orientations that could be found in a real molecule.

A search for potential ligands was then made by searching a database forknown molecular structures that match the positions of these groups ofsite points, using PLIM_DBS.

The best of these database hits were examined visually using acomputer-graphic modelling system, and the most promising of these wereselected according to a wealth of physico-chemical reasoning.

Modifications were made as described above to improve binding andsimplify synthesis. For example, phenyl rings were added to give bettercomplementarity to the hydrophobic surface of the binding site, chargedgroups were added to bind to charged groups on the protein that had beenoutside the scope of Plim, frequently toxic sub-structures were removed,and groups were replaced with functionally similar ones for syntheticreasons. Many such judgements were made, resulting in two families ofcompounds (hdo and bpy) with differing combinations of characteristics.

The efficacy of these modifications was finally assessed usingmolecular-dynamics free energy calculations as described herein to studythe stability of the protein-ligand complex (Åqvist et al., 1994).

Two structures from one family and one from the second family were thendeemed worthy of synthesis and testing.

The development of a number of the member of these families of compoundswill now be described in detail.

The first family (herein referred to as hdo) was derived from a databaseentry for 6-hydroxydopamine, denoted hdo_(—)0 (FIG. 8). The moleculebinds to the PapD side chains of Arg-8 and Lys-112, and to the backboneof Lys-110 through hydrogen-bond donation from the hydroxyl groups. Theprimary-amino group hydrogen-bonds to the side chain of Thr-170.

Derivative hdo_(—)1 (FIG. 8) was created from this base structure byreplacing an hydroxyl group with a carboxylic acid that can have acharge interaction with Arg-8 and Lys-112, and replacing the hydroxylpara to this position with a new phenyl ring, in order to fill out thehydrophobic cleft in the protein formed by the residues Ile-111, Leu-4and part of Thr-7. hdo_(—)4 (FIG. 8) was created from hdo_(—)1 byreplacing the original aromatic ring with a sugar, attaching thehydroxyl, primary amine and carboxylic acid substituents in such a wayas to maintain similar relative geometries, and then adding hydroxylgroups to one side of the new benzene ring so as to present ahydrophillic face to the solvent in this region.

At this point the structure looked convincing enough to invest time in amolecular-dynamics simulation, the results of which suggested that theamino group would not in fact bind to the gamma-carbon of Thr-7 of PapD,but would preferentially point into solution, satisfying itshydrogen-bonding requirements with water molecules rather than with theprotein side-chain. Thus, the amine group was replaced by the singlydonating hydroxyl group, a step that also simplified synthesis, theresult being structure hdo_(—)6 (FIG. 8).

Due to the perceived probability of toxicity due to the hydroxylsubstitution pattern on the benzene ring, the para-hydroxyl group wasremoved to give structure hdo_(—)7 (FIG. 8), and the remaining hydroxylgroup on this ring was replaced by a nitro group for further ease ofsynthesis (hdo_(—)8, FIG. 8).

A final compound with the previous nitro group left off was alsoconsidered a possibility, and was known as hdo_(—)9 (FIG. 8).

Another molecular-dynamics simulation was run, on hdo_(—)9, the resultsof which suggested that now the predicted interactions would indeed bemade and maintained in solution. A statistical analysis of thetrajectories with the program “Miss_Fit” showed that the conformation ofthe ligand did not change substantially during the run, and changes thatdid occur were compensated for by complementary shifts in the proteinstructure that maintained all significant interactions.

Dynamics simulation of hdo_(—)8 was complicated by the lack ofavailability of suitable molecular-mechanics parameters for themeta-nitro substituted ring, which made assessment throughmolecular-dynamics simulations harder to rely upon, and this influencedthe decision to pursue hdo_(—)9.

The range of possibilities that were considered during the design phaseof the hdo family are summarised in FIG. 9.

The second family, known as bpy, was derived from a database entry for(methyl-O,N,N-azoxy)-methyl-β-D-glucopyranoside cycasin (Kawaminami etal, 1981) denoted as bpy_(—)0 herein:

The conformation stored in the database was non-optic for binding to thePapD site, though it was close enough to be picked out by the Plim_DBSsearch. Small adjustments made to the internal torsion angles improvedthe binding pattern somewhat.

It is interesting to note that there are precedents for the approach ofusing a central carbohydrate as a scaffold to which functionalities areattached as to mimic a peptide (Hirschmann et al, 1992).

Derivative bpy_(—)1 was then created by adding a phenyl ring to the6-position, in order to fill out the hydrophobic cleft in the protein.The 4-OH group should form a favourable dipole interaction with theelectron-rich centre of the 6-phenyl group, and together with the 6-OH .. OS intramolecular hydrogen bond should stabilize the desired C5-C6rotamer.

It was decided that the Lys-112 and Arg-8 side chains that shouldinteract with the hydroxyl group at the 2-position in the ligand do soeven more with a charged group, and therefore bpy_(—)2 was created byreplacing this hydroxyl with a sulphate, bpy_(—)3 by replacing it with acyclic phosphate, and bpy_(—)5 by replacing it with a non-cyclicphosphate.

bpy_(—)4 was created by removing the 3-hydroxyl group from BPY_(—)2 sothat the sulphate group had a better chance of interacting with theArg-8 side chain, and bpy_(—)6 was analogously created by removing the3-hydroxyl group from bpy_(—)5. The following formula showsbpy_(—)1-bpy_(—)6:

R₁ R₂ —OH —OH bpy_1 —OH —OSO₂ ⁻ bpy_2 —O—PO₂ ⁻ —O— bpy_3 —H —OSO₂ ⁻bpy_4 —OH —OPO₂ ²⁻ bpy_5 —H —OPO₂ ²⁻ bpy_6

Consideration was then given to a small hydrophobic pocket and patchesor hydrophobic residues exposed to the solvent near position 1 on theligand. An aglycon that could make contact with these parts of theprotein and preferably also interact with solvent was sought. Theseconsiderations led to the replacement of the azoxy group at position 1with a 2-hydroxy-4-methylcyclopentyl group, resulting in bpy_(—)7:

At this point, a molecular dynamics run was made, the results of whichsuggested that the structure bound well, but that the expectedhydrophobic contacts between the cyclopentane and the protein were notmaintained.

Thus, bpy_(—)9 was created from bpy_(—)7 with the cyclopentane groupremoved, replaced by an α-methyl group for internal stability. Instead,further hydrophobic contact was attempted by the addition of an ethoxygroup axial on position 2. The charge was moved from equatorial 2 toaxial 3, a move stabilised by the inclusion of a NH-link that can donatea hydrogen bond to the oxygen at axial 1. The stereochemical arrangementof functionalities on positions 1-3 now confers conformational rigidityto the structure, as well as a simplification of synthesis.

More dynamics were run, this time suggesting that bpy_(—)9 would form astable complex with the protein, similar to the way that was envisagedduring the design.

Further Developments

By using reasoning in line with the above-described, more compounds havebeen designed (both from the hdo_family and from the bpy_family). Thecompounds of interest are those described by the general formulas I andII herein. The most promising compounds resulting from the analyses havebeen synthesized, cf. example 4.

In the further development of designed compounds using theabove-described methods it has interestingly been found that thestability of the binding between the designed compounds and PapD seemsto be only limitedly dependent on the interaction between the compoundand the Arg-8 residue, even though Arg-8 is essential for the in vivofunction of PapD. On the other hand, the stability of the interactionbetween the designed compounds and PapD is still very much dependent onthe interaction with Lys-112 as well as with the β-strand. Theseobservations confirm the surprising observation that the stable bindingbetween PapD and pilus subunits is very much dependent on theinteraction between the β-sheets of the two molecules.

Example 4

Synthesis of Compounds of the Hdo Family Capable of Binding to theBinding Site of PapD

General Methods

¹H- and ¹³C-NMR spectra were recorded in CDCl₃ at 300 and 75 MHzrespectively on a Varian Gemini 300 spectrometer unless otherwisestated. Signals from undeuterated solvent at 7.25 and 77.0 ppmrespectively were used as internal reference signals. Optical rotationswere measured using a Perkin Elmer 241 polarimeter.

Thin layer chromatography was performed on Merck DC-Fertigplatten(Kiselgel 60 F₂₅₄ 0.25 mm) and spots visualized by spraying with 10%sulphuric acid followed by charring at elevated temperature and/orspraying with molybdatophosphoric acid hydrate/Ce(SO₄)₂/dil. H₂SO₄followed by heating (molybdatophosphoric acidhydrate=H₃[P(Mo₃O₁₀)₄]×H₂O, E. Merck, Darmstadt, Germany)

Ethyl 2,3-O-dibenzoyl-4-O-benzyl-1-thio-β-D-glucohexopyranoside

Ethyl 2,3-O-dibenzoyl-4,6-O-benzylidene-1-thio-β-D-glucohexopyranoside(150 mg, 0.29 mmol) was dissolved together with borane-dimethylaminecomplex (68 mg, 1.16 mmol) in dry toluene (20 Å). Powdered 4 Å molecularsieves (180 mg) was added and the mixture was stirred at roomtemperature for 20 minutes. Aluminum trichloride (154 mg, 1.16 mmol) wasadded and after disappearance of the starting material (approx. 10 min.,silica tlc toluene/acetonitrile 4:1) the mixture was filtered, treatedwith Dowex(H⁺) ion-exchange resin until the solution was clear, andfiltered again. The filtrate was concentrated and co-evaporated twicewith methanol to yield 200 mg residue which upon chromatography onsilica (toluene-acetonitrile 8:1) gave 60 mg, 40% of ethyl2,3-O-dibenzoyl-4-O-benzyl-1-thio-β-D-glucohexopyranoside.

¹H-NMR (CDCl₃, δ): 8.0-7.2 (mm, 15H), 5.75 (t, 9.4 Hz, 1H, H3), 5.37 (t,9.8 Hz, 1H, H2), 4.74 (d, 9.8 Hz, 1H, H1), 4.61 (s, 2H), 4.02-3.95 and3.85-3.76 (2 bm, 2H, 2H6) 3.94 (t, 9.5 Hz, 1H, H4), 3.63 (ddd, 9.7 Hz,3.9 Hz, 2.6 Hz, 1H, H5), 2.74 (2q, 7.5 Hz, 2H), 1.25 (t; 7.5 Hz, 3H).

Ethyl6-O-acetyl-2,3-O-dibenzoyl-4-O-benzyl-1-thio-β-D-glucohexopyranoside

Ethyl 2,3-O-dibenzoyl-4-O-benzyl-1-thio-β-D-glucohexopyranoside (200 mg,0.38 mmol) was dissolved in an ice-cooled mixture of pyridine and aceticanhydride (1:1, 6 Å). The mixture was stirred and attained at roomtemperature overnight. The mixture was diluted with dichloromethane,washed with satd. aq. sodium hydrogen carbonate and water and dried oversodium sulphate. The solvents were removed under reduced pressure andthe residue was subjected to flash chromatography on silica gel(toluene-acetonitrile 6:1) to yield 204 mg, 93% of ethyl6-O-acetyl-2,3-O-dibenzoyl-4-O-benzyl-1-thio-β-D-glucohexopyranoside.

¹H-NMR (CDCl₃, δ): 8.0-7.2 (mm, 15H), 5.75 (t, 9.2 Hz, 1H, H3), 5.40 (t,9.8 Hz, 1H, H2), 4.71 (d, 10.0 Hz, 1H, H1) 4.59 and 4.55 (2d, 10.8 Hz,2H, bzl-CH₂), 4.44 (dd, 12.0 Hz, 1.9 Hz, 1H, H6), 4.27 (dd, 12.2 Hz, 4.6Hz, 1H, H6′) 3.87 (t, 9.5 Hz, 1H, H4), 3.77 (ddd, 9.7 Hz, 4.6 Hz, 1.9Hz, 1H, H5), 2.73 (m, 2H), 2.09 (s. 3H), 1.26 (t, 3H).

Methylglycolyl6-O-acetyl-2,3-O-dibenzoyl-4-O-benzyl-β-D-glucohexopyranoside

6-O-Acetyl-2,3-O-dibenzoyl-4-O-benzyl-1-thio-β-D-glucohexopyranoside(600 mg, 1.04 mmol) and N-iodosuccinimide (468 mg, 2.08 mmol, dried 3 h,<0.1 mbar) were dissolved in dry acetonitrile (7 Å, passed throughalumina) at room temperature. Methyl glycolate (161 μl, 2.08 mmol) wasadded and the mixture was stirred for 25 min at room temperature andthen cooled on an ice-bath. Trifluoromethylsulphonic acid (18 μl, 0.21mmol) was carefully added. Complete conversion of starting material hadoccurred after 10 minutes (tlc: silica toluene-acetonitrile 4:1). Thereaction was quenched by addition of triethylamine, the mixture wasdiluted with dichloromethane, washed with water, sodium hydrogencarbonate and dried over sodium sulphate. The solvents were removedunder reduced pressure and the residue subjected to chromatography onsilica (toluene-acetonitrile 11:1) to yield 560 mg, 86% of ethyl6-O-acetyl-2,3-O-dibenzoyl-4-O-benzyl-1-thio-β-D-glucohexopyranoside.

Ethyl 2,3-O-Dibenzoyl-4-O-benzyl-1-thio-β-D-glucohexopyranoside(hdo_(—)9:1)

Ethyl 2,3-O-dibenzoyl-4,6-O-benzylidene-1-thio-β-D-glucohexopyranoside(Chemical Abstracts, RN 149563-08-6 or Hällgren and Widmalm, 1993) wasdissolved together with borane-dimethylamine complex (68 mg, 1.16 mmol)in dry toluene (20 ml) (reaction “a” in FIG. 6). Powdered 4 Å molecularsieves (180 mg) were added and the mixture was stirred at roomtemperature for 20 minutes. Aluminium trichloride (154 mg, 1.16 mmol)was added and after disappearance of the starting material (approx. 10min., silica tlc toluene/acetonitrile 4:1) the mixture was filtered,treated with Dowex(H⁺) ion-exchange resin until the solution was clear,and filtered again. The filtrate was concentrated and co-evaporatedtwice with methanol to yield 200 mg residue which upon chromatography onsilica (toluene-acetonitrile 8:1) gave 60 mg, 40% of hdo_(—)9:1.

¹H-NMR (CDCl₃, δ): 8.0-7.2 (mm, 15H), 5.75 (t, 9.4 Hz, 1H, H3), 5.37 (t,9.8 Hz, 1H, H2), 4.74 (d, 9.8 Hz, 1H, H1), 4.61 (s, 2H), 4.02-3.95 and3.85-3.76 (2 bm, 2H, 2H6) 3.94 (t, 9.5 Hz, 1H, H4), 3.63 (ddd, 9.7 Hz,3.9 Hz, 2.6 Hz, 1H, H5), 2.74 (2q, 7.5 Hz, 2H), 1.25 (t; 7.5 Hz, 3H).

Ethyl6-O-acetyl-2,3-O-dibenzoyl-4-O-benzyl-1-thio-β-D-glucohexopyranoside(hdo_(—)9:2)

Ethyl 2,3-O-dibenzoyl-4-O-benzyl-1-thio-β-D-glucohexopyranoside(hdo_(—)9:1) (200 mg, 0.38 mmol) was dissolved in an ice-cooled mixtureof pyridine and acetic anhydride (1:1, 6 ml). The mixture was stirredand attained room temperature overnight. The mixture was diluted withdichloromethane and washed with saturated aqueous sodium hydrogencarbonate and water, dried over sodium sulphate. the solvents wereremoved under reduced pressure and the residue was subjected to flashchromatography on silica gel (toluene-acetonitrile 6:1) to yield 204 mg,93% of hdo_(—)9:2.

¹H-NMR (CDCl₃, δ): 8.0-7.2 (mm, 15H), 5.75 (t, 9.2 Hz, 1H, H3), 5.40 (t,9.8 Hz, 1H, H2), 4.71 (d, 10.0 Hz, 1H, H1) 4.59 and 4.55 (2d, 10.8 Hz,2H, bzl-CH2), 4.44 (dd, 12.0 Hz, 1.9 Hz, 1H, H6), 4.27 (dd, 12.2 Hz, 4.6Hz, 1H, H6′) 3.87 (t, 9.5 Hz, 1H, H4), 3.77 (ddd, 9.7 Hz, 4.6 Hz, 1.9Hz, 1H, H5), 2.73 (m, 2H), 2.09 (s. 3H), 1.26 (t, 3H).

Methylglycolyl6-O-acetyl-2,3-O-dibenzoyl-4-O-benzyl-β-D-glucohexopyranoside(hdo_(—)9:3)

6-O-acetyl-2,3-O-dibenzoyl-4-O-benzyl-β-thio-β-D-glucohexopyranoside(hdo_(—)9:2) (600 mg, 1.04 mmol) and N-iodosuccinimide (468 mg, 2.08mmol, dried 3 h, <0.1 mbar) were dissolved in dry acetonitrile (7 ml,passed through alumina) at room temperature. Methyl glycolate (161 μl,2.08 mmol) was added and the mixture was stirred for 25 min at roomtemperature and then cooled on an ice-bath. Trifluoromethylsulphonicacid (18 μl, 0.21 mmol) was carefully added. Complete conversion ofstarting material had occurred after 10 minutes (tlc: silicatoluene-acetonitrile 4:1). The reaction was quenched by addition oftriethylamine and the mixture was diluted with dichloromethane, washedwith water, sodium hydrogen carbonate and dried over sodium sulphate.The solvents were removed under reduced pressure and the residuesubjected to chromatography on silica (toluene-acetonitrile 11:1) toyield 560 mg, 86% of (hdo_(—)9:3).

Sodium Glycolyl 4-O-benzyl-β-D-glucohexopyranoside (hdo_(—)9)

Methylglycolyl6-O-acetyl-2,3-O-dibenzoyl-4-O-benzyl-β-D-glucohexopyranoside(hdo_(—)9:3) (350 mg, 0.59 mmol) was dissolved in 7 Å 25 mM sodiummethoxide (methanol) and stirred at room temperature for 20 h or untilonly a very slow-moving spot could be detected on tlc by sulphuric acidcharring (silica, toluene-acetonitrile 4:1). A small sample of thissolution (=i) was retained as tlc reference before the methyl glycolatehydrolysis. Sodium hydroxide (15 mg) and water (200 μl) was added andstirring was continued for 8 h or until all “i” was consumed (tlcsilica, ethyl acetate-ethanol-acetic acid 8:3:1). The reaction wasquenched by bringing the pH to 4.2 with acetic acid. The solvents wereremoved under reduced pressure and the residue (650 mg) was subjected toflash chromatography on silica (ethyl acetate-ethanol-acetic acid8:3:1). The material in the R_(f)-range 0.15-0.25 was collected andconcentrated to yield 178 mg, 92% of amorphous white solid.

¹H-NMR (CD₃OD, δ relative to methanol at 3.31 ppm): 7.45 (m, 5H), 4.65(d, 11.0 Hz, 1H), 4.36 (d, 16.0 Hz, 1H), 4.32 (d, 7.9 Hz, 1H, H1), 4.11(d, 15.8 Hz, 1H), 3.82 (dd, 2.0 Hz, 12.1 Hz, 1H, H6), 3.65 (dd, 4.8 Hz,12.1 Hz, 1H, H6), 3.59 (t, 9.0 Hz, 1H, H3 or H4), 3.40 (t, 9.1 Hz, 1H,H3 or H4)

This material still contained much acetic acid, so 50 mg was redissolvedin water and freeze-dried to give 30 mg of substance free acetic acidbut with a water content (¹H-NMR) of 24.5 mol. eq.

This material was again redissolved in water and 1 equivalent of sodiumhydroxide was added, the solution was freeze-dried to give 22 mg of thetitle compound.

¹H-NMR (CD₃OD, δ relative to methanol at 3.31 ppm): 7.40-7.22 (m, 5H),4.95 (d, 10.8 Hz, overlap by residual HDO at 4.93), 4.65 (d, 11.0 Hz,1H), 4.34 (d, 16.0 Hz, 1H), 4.32 (d, 7.5 Hz, 1H, H1), 4.10 (d, 15.8 Hz),3.82 (dd, 1.8 Hz, 11.9 Hz, 1H, H6), 3.66 (dd, 4.8 Hz, 12.1 Hz, 1H, H6),3.59 (t, 8.8 Hz, 1H, H3), 3.41 (t, 9.7 Hz, 1H, H4), 3.15-3.06 (m, 2H,H5, H2).

¹³C-NMR (CD₃OD, δ relative to methanol at 49.0 ppm): 176.1, 140.0,129.3, 129.1, 128.6, 104.3, 79.1, 78.1, 77.2, 75.7, 75.2, 68.4, 62.3.

2-(Hydroxy)ethyl 4-O-benzyl-β-D-glucopyranoside (hdo_(—)23)

A mixture of N-Iodosuccinimide (0.109 g, 0.484 mmol) and triflic acid(6.6 μl, 0.0744 mmol) in dichloroethane-diethyl ether (2 ml, 1:1) wasadded to a stirred mixture of the thioglycoside ethyl6-O-acetyl-2,3-O-benzoyl-4-O-benzyl-1-thio-β-D-glucopyranoside(hdo_(—)9:2) (0.210 g, 0.372 mmol), ethylen glycol (0.125 ml, 2.232mmol), and molecular sieves (0.3 g, 4 Å) in dichloromethane-diethylether (3 ml, 1:2) at room temperature during 5 min. After 25 min TLCshowed approx. 20% conversion of the thioglycoside, thus moreN-Iodosuccinimide (0.109 g, 0.484 mmol) and triflic acid (6.6 μl, 0.0744mmol) in dichloroethane-diethyl ether (2 ml, 1:1) was added during 5min. After additional 30 min. the reaction mixture was filtered througha layer of Celite into an aqueous solution of sodium hydrogen carbonateand sodium bisulphite. The organic layer was separated, washed withwater, dried (Na₂SO₄) and concentrated. The residue was coevaporetedtwice with toluene, dissolved in methanol containing sodium methoxide(10 ml, 0.2 M), kept 1 h at 50° C., and evaporated. Columnchromatography (SiO₂, chloroform-methanol-water, 100:15:1) of theresidue gave hdo_(—)23 (33 mg, 30%

[α]_(D) ²²+0.9° (c 1.4, methanol)

TLC: Rf 0.29 (chloroform-methanol-water, 100:15:1).

¹³C-NMR (CD₃OD, δ: relative to methanol at 49.0 ppm) δ: 139.9, 129.2,129.0, 128.6, 104,4 (C-1), 79.2, 78.2, 77.0, 75.7, 75.4, 72.3, 62.3,.62.3 ppm.

¹H-NMR (CD₃OD, δ: relative to methanol at 3.31 ppm)) δ: 7.45-7.25 (m,5H), 4.95 (d, 11.0 Hz, 1H, beazylic),4.65 (d, 11.0 Hz, 1H, benzylic),4.29 (d, 7.7, Hz, 1H, H-1) ppm.

Example 5

Synthesis of Compounds of the Bpy Family Capable of Binding to theBinding Site of PapD

Methyl 4′-methoxyphenylmethylene-α-D-mannohexopyranosides, Mixture of4,6-, 2,3-endo and 2,3-exo Monoacetals

(See: Patroni et al, 1988).

Methyl α-D-mannohexopyranoside (38.8 g, 200 mmol) and p-toluenesulphonicacid (200 mg) were dissolved in dimethylformamide (300 ml) and stirredat 100° C. under a nitrogen stream. 4-methoxy-benzaldehyde dimethylacetal (37.7 g, 240 mmol) in dimethylformamide (300 ml) was addeddropwise under 3 h. Since starting material still remained (tlc, silica,ethyl acetate), stirring and heating was continued for 2 h. Thecomposition of the reaction mixture did not show any visible change atthat time (tlc, silica, ethyl acetate) and the reaction was quenchedwith potassium carbonate (2.15 g). The solvent was removed under reducedpressure and the residue was filtered through silica (ethyl acetate) toremove salts and starting material. The concentrated eluents depositedmonoacetals (¹H-NMR) on trituration with methyl t-butyl ether (11 g,18%), which was collected by filtration. The filtrates were subjected toflash chromatography to yield additional 20 g of the monoacetal mixture(yield 51) and 17 g of the 2,3;4,6-di acetals (endo/exo≈1:1).

Methyl2-O-ethyl-4,6-O-(4′-methoxy)phenylmethylene-α-D-mannohexopyranoside(bpy_(—)9:1)

Methyl 4′-methoxyphenylmethylene-α-D-mannohexopyranosides, mixture of4,6-, 2,3-endo and 2,3-exo monophenylmethylene acetals (11.3 g, 36.2mmol), ethyl iodide (4.35 ml; 54.3 mmol) and tetrabutylammonium hydrogensulphate (2.48 g, 7.24 mmol) were dissolved in methylene chloride (500ml). To this was added a solution of sodium hydroxide (2.5 g, 140 mmol)in water (50 ml) and the mixture was heated at reflux for four days,with daily additions of ethyl iodide (2.4 ml) and 20% sodium hydroxide(1 ml). After this time, no visible change in the composition of thereaction mixture could be defected by tlc (toluene-ethyl acetate 2:1).The mixture was allowed to cool, the phases separated and the organicphase washed with water (3×) and dried over sodium sulphate. The solventwas removed under reduced pressure and the residue was subjected toflash chromatography on silica gel (toluene-ethyl acetate 6:1). Thefractions containing the component with R_(f)=0.40 on tlc (toluene-ethylacetate 2:1) were pooled and recromatographed as above to yield 3.15 g,26% of the title compound. Fractions containing components withR_(f)=0.32 on tlc were found to consist of a mixture of monoethylatedmonophenylmethylene acetal regio-isomers (¹H-NMR).

[α]_(D) ²⁰=+11.2° (c1.02, CHCl₃)

¹H-NMR (CDCl₃, δ): 7.42 (symm.m, 2H), 6.88 (symm.m, 2H), 5.53 (s, 1H),4.77 (d, 1.3 Hz, 1H, H1), 4.24 (dd, 4.2 Hz, 9.7 Hz, 1H, H6), 4.02 (dt,4.0 Hz, 8.8 Hz, 1H, H5), 3.83 (dd, 1.8 Hz, 9.7 Hz, 1H, H6), 3.80-3.69 (sand overl.m, 6H, 1-OMe, H3,H4, 2-O-CHHCH₃), 3.66 (dd, 1.3 Hz, 3.7 Hz,1H, H2), 3.64-3.55 (m, 1H, 2-O-CHHCH₃), 3.69 (s, 3H, Ar-OMe), 2.48 (d,1H, OH), 1.26 (t, 7.0 Hz, 3H, 2-O-CH₂CH ₃).

¹H-NMR (CDCl₃, with added Cl₃CCONC, δ): 8.54 (s, 1H, acylcarbamate NH),7.38 (symm.m, 2H), 6.88 (symm.m, 2H), 5.52 (s, 1H), 5.27 (dd, 3.5 Hz,10.3 Hz, 1H, H3), 4.76 (d, 1.6 Hz, 1H, H1), 4.32-4.20 (m, 1H), 4.13(symm.m, 1H, H5), 3.97 (dd, 1.8 Hz, 3.5 Hz, 1H, H2), 3.91-3.80 (m, 1H),3.79 (s, 3H, 1-OMe), 3.77-3.54 (m, 3H), 3.41 (s, 3H, Ar-OMe), 1.23 (t,7.0 Hz, 3H).

¹³C-NMR (CDCl₃, δ): 160.1, 129.9, 127.6, 113.6, 102.0, 99.3, 79.4, 78.8,68.7, 68.3, 67.2, 63.2, 55.2, 54.9, 15.4.

Calc. for C₁₇H₂₅O₇: C: 59.8 H: 7.38,

Methyl2-O-ethyl-3-O-dimethyl-t-butylsilyl-4,6-O-(4′-methoxy)phenylmethylene-α-D-mannohexopyranoside(bpy_(—)9:2)

Methyl2-O-ethyl-4,6-O-(4′-methoxy)phenylmethylene-α-D-mannohexopyranoside(bpy_(—)9:1) (0.98 g, 2.88 mmol) was dissolved in dry methylene chloride(18 ml) together with triethyl amine (600 μl, 4.32 mmol) and the mixturewas ice-cooled. t-Butyl-dimethylsilyl trifluromethyl sulphonate (795 μl,3.45 mmol) in methylene chloride (2 ml) was added dropwise and thereaction mixture with the cooling bath was left at ambient temperatureovernight (tlc silica toluene-methyl t-butyl ether 4:1). The excesssilylating reagent was destroyed by addition of methanol (3 ml), themixture was concentrated under reduced pressure and the residue wassubjected to flash chromatography on silica (toluene-methyl t-butylether 40:1) to yield 1.40 g, 100% of (bpy_(—)9:2).

¹H-NMR (CDCl₃, δ): 7.40 (symm.m, 2H), 6.87 (symm.m, 2H), 5.53 (s. 1H),4.70 (d, 1.3 Hz, 1H, H1), 4.21 (dd, 4.6 Hz, 9.9 Hz, 1H, H6), 4.10 (dd,3.3 Hz, 9.7 Hz, 1H, H3), 3.91 (t, 9.5 Hz, 1H, H4), 3.90-3.79 (s overl.m, 5H, H6, 2-OCHHCH₃;1-OMe), 3.76-3.63 (m, 2H, H5, 2-OCHHCH₃), 3.55 (dd,1.5 Hz, 3.3 Hz, 1H, H2), 3.67 (s, 3H, Ar-OMe), 1.24 (t, 7.0 Hz, 3H),0.88 (s, 9H), 0.08 (s, 3H), 0.03 (s, 3H)

¹³C-NMR (CDCl₃, δ): 159.9, 130.3, 127.5, 113.4, 101.8, 101.2, 79.9,79.1, 70.3, 68.8, 68.2, 64,1, 55.2, 54.8, 25.8, 18.3, 15.6, −4.4, −4.9.

Methyl2-O-ethyl-3-O-dimethyl-t-butylsilyl-4,-O-(4′-methoxy)benzyl-α-D-mannohexopyranoside(bpy_(—)9:3) and Methyl2-O-ethyl-3-O-dimethyl-t-butylsilyl-6-O-(4′-methoxy)benzyl-α-D-mannohexopyranoside(bpy_(—)9:8)

Methyl2-O-ethyl-3-O-dimethyl-t-butylsilyl-4,6-O-(4′-methoxy)phenylmethylene-α-D-mannohexopyranoside(bpy_(—)9:2) (3.61 g, 7.95 mmol) was dissolved in acetonitrile (150 ml,dried over 4 Å molecular sieves), powdered 3 Å molecular sieves (6 g)was added and the mixture cooled to −30° C. under a nitrogen atmosphereand stirred for 15 min. Sodium cyanoborohydride (3.04 g, 47.7 mmol) wasadded and a solution of chlorotrimethylsilane (7.95 ml, 47.7 mmol) indry acetonitrile (50 ml, dried over 4 Å molecular sieves) was addeddropwise in 1 h. Stirring was continued for 2 h at −30° C., whereafterthe temperature was allowed to rise to 0° C. After 1 h tlc(toluene-ethyl acetate 4:1) showed complete conversion of startingmaterial. The mixture was filtered through a Celite® pad, the Celite®washed thoroughly with ethyl acetate. The combined filtrates were washedwith aq. satd. sodium hydrogen carbonate, aq. satd. sodium chloride,concentrated under reduced pressure, redissolved in toluene andconcentrated under reduced pressure. The residue was subjected to flashchromatography on silica (toluene-ethyl acetate 4:1, then 1:1) to obtainbpy_(—)9:8 (=bpy_(—)21:1) 578 mg, 16% and (bpy_(—)9:3) 2.34 g, 65%.

bpy_(—)9:3 (Some Assignments From COSY, HETCOR)

¹H-NMR (CDCl₃, δ): 7.25 (symm.m 2H), 6.87 (symm.m, 2H), 4.81 (d, 11.0Hz, 1H), 4.66 (d, 1.8 Hz, 1H, H1), 4.51 (d, 10.8 Hz, 1H), 4.02 (dd, 3.1Hz, 5.9 Hz, 1H, H3), 3.80 (s, 3H, 1-OMe), 3.79-3.60 (m, 5H, H4, 2H6,2-OCH ₂CH₃), 3.54 (ddd, 2.9 Hz, 4.8 Hz, 9.7 Hz, 1H, H5), 3.46 (dd, 2.0Hz, 3.3 Hz, 1H, H2), 3.33 (s, 3H, Ar-OMe), 2.10 and 1.86 (2 v.br.s, ≈1H,6-OH), 1.23 (t, 6.8 Hz, 3H), 0.95 (s, 9H), 0.12 (s 6H).

¹H-NMR (CDCl₃, with added Cl₃CCONC, δ) 8.40 (s, 9H, acylcarbamate NH),7.24 (symm.m, 2H, Ar H3′, 5′), 6.86 (symm.m, 2H, Ar H2′, H6′), 4.82 (d,11.2 Hz, 1H), 4.67 (d, 2.0 Hz, 1H, H1), 4.43-4.32 (m₁ 2H, 2H6),4.08-3.98 (m, 1H, H3), 3.78 (s, 3H, 1-OMe), 3.77-3.61 (m, 4H, H5, 2-OCH₂CH₃, H4), 3.46 (dd, 1.8 Hz, 3.1 Hz, 1H, H2), 3.34 (s, 3H, Ar-OMe), 1.22(t, 7.0 Hz, 3H), 0.96 (s, 9H), 0.14 (s, 3H), 0.13 (s, 3H).

¹³C-NMR (CDCl₃, δ): 159.1, 130.7, 129.3, 113.8, 99.8, 79.5, 75.7, 74.7,73.0, 72.2, 67.4, 62.5, 55.2, 54.7, 18.0, 15.7, −4.3, −4.6.

¹³C-NMR (CDCl₃, with added Cl₃CCONC, δ): 159.3 (Ar C4′), 157.4, 149.3,130.2 (Ar C2′), 129.6 (Ar C3′,C5′), 113.8 (Ar C2′, C6′), 99.6 (C1), 79.2(C2), 74.56*(4-OCH₂Ar or 2-CH₂CH₃), 74.54*(4-OCH₂Ar or 2-CH₂CH₃), 73.2(C3), 69.7 (C5), 67.2 (C4), 66.3 (C6), 55.2 (1-OMe), 54.9 (Ar-OMe), 25.9(CMe ₃), 18.0 (CMe₃), 15.6, −4.3, −4.7.

bpy_(—)9:8

¹H-NMR (CDCl₃, δ): 7.28 (symm.m, 2H), 6.86 (symm.m, 2H), 4.70 (d, 1.8Hz, 1H, H1), 4.55 (d, 11.7 Hz, 1H, H6), 4.51 (d, 11.6 Hz, 1H, H6),3.89-3.55 and 3.79 (m and s, 7H, H3, H4, H5, 2-OCH ₂CH₃, 1-OMe), 3.44(dd, 1.8 Hz, 2.9 Hz, 1H, H2), 3.56 (s, 3H, Ar-OMe), 2.29 (d, 2.0 Hz, 1H,4-OH), 1.20 (t, 7.0 Hz, 3H), 0.92 (s, 9H), 0.13 (s, 3H), 0.12 (s, 3H).

¹³C-NMR (CDCl₃, δ): 159.1, 130.3, 129.3, 113.7, 99.7, 78.9, 73.19*,73.16*, 71.3, 70.3, 69.1, 67.2, 55.2, 54.8, 25.8, 18.2, 15.6, −4.5,−4.6.

*resolved by multiplying the FID with a gaussian weigth-function.

Methyl 2-O-ethyl-3-O-dimethyl-t-butylsilyl-4,-O-(4′-methoxy)benzyl-6(S)-phenyl-α-D-mannohexopyranoside (bpy_(—)9:4)

(Oxidation ref. D. F. Taber et al, J. Org. Chem. 52, 5621-2, 1987)Methyl2-O-ethyl-3-O-dimethyl-t-butylsilyl-4,-O-(4′-methoxy)benzyl-α-D-mannohexopyranoside(bpy_(—)9:3) (200 mg, 0.438 mmol) and dimethylsulphoxide (64.0 μl, 0.876mmol) were dissolved in dichloromethane (2.5 ml, dried over 4 Åmolecular sieves) and the solution was cooled on an ice-bath.Phosphorpentoxide (124 mg, 0.876 mmol) was added quickly and theresulting stirred suspension was allowed to attain RT (1 h). The mixturewas again ice-cooled and triethyl amine (215 μl, 1.53 mmol) was addedcausing an immediate dissolution of the gel-like suspension. Theice-bath was removed and the mixture was stirred at RT for 1 h, thediluted with ethyl acetate, washed with aq. 0.2 M sodium dihydrogenphosphate, dried over sodium sulphate and concentrated under reducedpressure (<0.02 mbar). The residue was dissolved tetrahydrofurane (2 ml,dried over 4 Å molecular sieves), stirred and cooled to −20° C.Phenylmagnesium chloride in tetrahydrofurane (345 μl of a 25 w %solution, 0.657 mmol, Janssen, Belgium) was added dropwise (3 min). Thereaction was monitored with tlc (toluene-ethyl acetate 4:1) and quenchedwith aq. 10% ammonium sulphate (3 ml). The mixture diluted with ethylacetate and washed twice with water, dried over sodium sulphate andconcentrated under reduced pressure. The residue was subjected to flashchromatography on silica (toluene-methyl t-butyl ether 20:1). Thefractions containing the component with a R_(f)=0.44 on tlc(toluene-ethyl acetate 4:1) were pooled and concentrated under reducedpressure to yield 65 mg, 30% of the title compound.

Molecular mechanics calculations with MM2(91) (Burkert and Allinger,1982) of the R and S diastereomers and subsequent calculations withKarplus equation (Bothner-By, 1965) predicted that the ¹H-NMR J₅₋₆ wouldbe 4.8 and 6.2 respectively. This difference is too small to be but anindication of the stereochemistry. Furthermore, the H6 signal is poorlyresolved and the H5 signal is severely overlapped by other resonances.The ¹H-COSY H4-H5 crosspeak, however, contains the sum J₄₋₅+J₅₋₆ andsince the J₄₋₅ is observable in the H4-resonance, the J₅₋₆ can beassigned the value 6.8 Hz. This indicates that the isolated product hasthe 6-(S) stereochemistry.

After the removal of the 4-methoxybenzyl group the calculated couplingconstants become 5.5 Hz and 12.9 Hz, respectively.

¹H-NMR (CDCl₃, δ): 7.44-7.19 (m, 7H), 6.89 (symm.m, 2H), 5.00 (br.m, 1H,H6), 4.90 (d, 10.8 Hz, 1H), 4.66 (d, 10.8 Hz, 1H), 4.58 (d, 1.8 Hz, 1H,H1), 4.04 (dd, 3.1 Hz, 9.0 Hz), 3.99 (t, 9.2 Hz, 1H, H4), 3.81 and3.82-3.56 (s and m, 6H, 1-OMe, 2-CH ₂CH₃), H5), 3.44 (dd, 2.0 Hz, 2.9Hz, 1H, H2), 2.84 (s, 3H, Ar-OMe), 1.24 (t, 3H), 1.97 (s, 9H), 0.15 (s,3H), 0.13 (s, 3H).

¹³C-NMR (CDCl₃, δ): 159.0, 142.5, 130.8, 129.2, 127.7, 126.7, 126.6,125.7, 113.7, 99.5, 79.8, 76.1, 74.5, 73.0, 70.9, 67.9, 55.1, 54.1,25.8, 17.9, 15.4, −4.5, −4.8.

Methyl 2,3-anhydro-4,6-O-p-methoxybenzylidene-α-D-mannopyranoside(bpy_(—)9:9)

To a stirred suspension of sodium hydride (2.6 g, 65 mmol, 65%dispersion in mineral oil) in N,N-dimethylformamide (150 ml) was addedwith stirring a solution of methyl4,6-O-p-methoxybenzylidene-α-D-glucopyranoside (9.36 g, 30 mmol) inN,N-dimethylformamide (65 ml). The mixture was stirred to 45 minutes andp-toluenesulfonylimidazol (7.24 g, 33 mmol was added. Stirring wascontinued for 2 hours and the mixture was the poured into ice-water. Theprecipitate was filtered of, dried in vacuo to give crude bpy_(—)9:)(7.7 g). Recrystallization twice form methanol (250 ml gave bpy_(—)9:9(3.58 g). Chromatography (SiO₂, ethyl acetate-heptane, 2:3) of themother liquor followed by crystallization form methanol (150 ml) gaveadditional bpy_(—)9:9 (1.87 g). The total yield of bpy_(—)9:9 was 5.45 g(61%).

m.p. 152-153.5° C. (methanol)

[α]_(D) ²²: 96.4° (c 1.8, chloroform)

¹H NMR (CDCl₃) δ: 7.43 and 6.91 (AB pattern, further coupled, 2H,J_(AB)=8.7 Hz), 5.53 (s, 1H, ArCH), 4.90 (s, 1H, H-1), 4.30-4.19 (sym.m,1H) 3.82 (s, 3H, CH₃OAr), 3.78-3.64 (3H), 3.49-3.46 (4H), 3.48 (s, 3H,CH₃O), 3.18 (d, 1H, J 3.7=Hz, H-2/H-3) ppm.

¹³C NMR (CDCl₃) δ: 160.3, 129.6, 127.5, 113.7, 102.4, 96.9, 74.8, 69.4,61.7, 55.7, 55.3, 53.8 and 50.5 ppm.

Methyl 3-azido-4,6-O-p-methoxybenzylidene-α-D-altropyranoside(bpy_(—)9:10)

Methyl 2,3-anhydro-4,6-O-p-methoxybenzylidene-α-D-mannopyranoside(bpy_(—)9:9, 2.35 g, 8 mmol), sodium azide (2.08 g, 32 mmol) andammonium chloride (0.86 g, 16 mmol) was stirred for 5 hours at 110° C.in a mixture of 2-methoxyethanol (25 ml) and water (5 ml). Thesuspension gradually dissolved to give a turbid solution. The mixturewas then partitioned between ethyl acetate and aqueous NaOH (0.25 M, 100ml). The aqueous phase was extracted twice with ethyl acetate and thecombined organic phases was washed twice with water and than withsaturated aqueous NaCl, dried (Na₂SO₄), filtered and concentrated.Chromatography (SiO₂) ethyl acetate-heptane, 2:3-2:1) gave bpy_(—)9:10(1.97 g, 73%). An analytical sample was crystallized from ethylacetate-heptane.

m.p. 91-93° C. (EtOAc/Heptane)

[α]_(D) ²²: +26.1° (c 1.1, chloroform)

¹H NMR (CDCl₃) δ: 7.44 and 6.91 (AB pattern further coupled, 2H,J_(AB)=8.6 Hz), 5.57 (s, 1H, ArCH), 4.56 (s, 1H, H-1), 4.43-4.21 (2H),4.16-4.08 (1H), 4.03 (unresolved dd, 1H, J₁=J₂=3 Hz), 3.91 (bs, 1H,H-2), 3.83-3.74 (4H), 3.80 (s, 3H, CH₃O); 3.43 (s, 3H, CH₃O), 2.36 (bs,1H, OH) ppm.

¹³C NMR (CDCl₃) δ: 160.3, 129.5, 127.5, 113.7, 102.3, 101.4, 75.8, 69.8,69.0, 60.1, 59.0,55.7 and 55.3 ppm.

Acetylation (acetic anhydride-pyridine, 3:5) of a sample of bpy_(—)9:10gave an acetate which had the following ¹H NMR spectrum (assignmentswere confirmed by the corresponding homonuclear COSY spectrum):

¹H NMR (CDCl₃) δ: 7.43 and 6.91 (AB pattern further coupled, 2H,J_(AB)=8.8 Hz), 5.59 (s, 1H, ArCH), 4.97 (dd, 1H; J=2.2 and 0.9 Hz,H-2), 4.57 (s, 1H, H-1), 4.36 (2H, H-6 and H-4), 4.09-4.01 (2H, H-6, andH-3), 3.84-3.74 (2H, CH₃O and H-5), 3.81 (s, 3H, CH₃O), 3.44 (s, 3H,CH₃O), 2.15 (s, 3H, CH₃CO) ppm.

Methyl 3-azido-2-O-ethyl-4,6-O-p-methoxybenzylidene-α-D-altropyranoside(bpy_(—)9:11)

Methyl 3-azido-4,6-O-p-methoxybenzylidene-α-D-altropyranoside(bpy_(—)9:10, 1.7 g, 5 mmol), barium oxide (3.0 g, 19.6 mmol) and ethyliodide (5 ml, 62 mmol) was stirred in dimethyl sulphoxide (5 ml). Water(10 μl) was added and the stirring was continued for 16 hours. Themixture was then partitioned between ethyl acetate and water. Theaqueous phase was extracted twice with ethyl acetate and the combinedorganic phases was washed twice with water and then with saturatedaqueous NaCl, dried (Na₂SO₄), filtered and concentrated. Chromatography(SiO₂, ethyl acetate-heptane, 1:3) gave bpy_(—)9:11 as an oil (1.7 g,92%).

[α]_(D) ²²: 19.1° (c 1.6, chloroform).

¹H NMR (CDCl₃) δ: 7.44 and 6.91 (AB pattern, further coupled, 2H,J_(AB)=8.6 Hz), 55.57 (s, 2H, ArCH), 4.63 (bs, 1H, H-1), 4.14-4.06 (2H),3.85-3.75 (4H), 3.81 (s, 3H, CH₃O), 3.73-3.56 (sym.m.2H), 3.52 (dd, 1H,J=2.4 and 0.9 Hz, H-3), 3.34 (s, 3H, CH₃O), 1.25 (t, 3H, J=6.9 Hz, CH₃CH₂) ppm.

¹³C NMR (CDCl₃) δ: 160.1, 129.6, 127.4, 113.6, 102.1, 99.6, 77.0, 76.1,69.0, 66.5, 58.7, 58.1, 55.5, 55.2 and 15.3 ppm.

Methyl3-azido-3-deoxy-2-O-ethyl-4-O-p-methoxybenzyl-δ-D-altro-pyranoside

(Cf. R. Johansson and B. Samuelsson; J. Chem. Soc. Commun. 1984,201-202).

Methyl3-azido-3-deoxy-2-O-ethyl-4,6-O-p-methoxybenzylidene-α-D-altropyranoside(bpy_(—)9:11) (2.41 g, 6.6 mmol), sodium cyanoborohydride (2.5 g, 39.6mmol) and ground molecular sieves (5 g) was stirred in acetonitrile (130ml) at 0° C. A solution of trimethylsilyl chloride (5.0 ml, 40 mmol) inacetonitrile (45 ml) was added during 55 min. After additional 1.5 h ofstirring, the cooling bath was removed and the stirring was continuedfor 21 h. The mixture was filtered through celite and the filtrate waspartitioned between ethyl acetate and saturated aqueous NaHCO₃. Theorganic phase was washed twice with saturated aqueous NaHCO₃ and thenwith saturated aqueous NaCl, dried (Na₂SO₄+NaHCO₃), filtered andevaporated. Chromatography twice (SiO₂, ethyl acetate-heptane 2:7→2:1and ethyl acetate-toluene 1:2→2:1) gave the title compound (2.23 g, 92%)as an oil.

[α]_(D) ²²: +113.6° (c 1.4, chloroform)

¹H NMR data (CDCl₃) δ: 7.30 and 6.89 (AB pattern, further coupled, 4H,J=8.8 Hz), 4.62 and 4.56 (AB pattern, 2H, J=11.3 Hz, benzylic H), 4.58(unresolved d, virtually coupled, 1H, J <2 Hz, H-1), 4.00-3.84 (3H, H-3,H-4 and H-5), 3.81 (s, 3H, CH₃OAr), 3.84-3.7 (2H, H-6), 3.62-3.43 (3H,CH ₂CH₃ and H-2), 3.38 (s, 3H, CH₃O), 1.99 (bs, 1H, OH) and 1.19 (t, 3H,J=7.0 Hz, CH₂CH ₃) ppm.

¹³C NMR data (CDCl₃) δ: 159.6, 129.9, 129.6, 113.9, 99.9, 76.9, 72.2,71.9, 67.7, 66.3, 62.4, 58.6, 55.4, 55.3 and 15.4 ppm.

Methyl3-azido-6-O-benzoyl-3-deoxy-2-O-ethyl-4-O-p-methoxybenzyl-α-D-altropyranoside

A solution of methyl3-azido-3-deoxy-2-O-ethyl-4-O-p-methoxybenzyl-α-D-altro-pyranoside (1.47g, 4 mmol) in pyridine (35 ml) was stirred at 0° C. Benzoyl chloride(1.2 ml, 10.3 mmol) was added and the temperature was raised to roomtemperature. The stirring was continued for 1.5 h and the mixture wasthen cooled to 0° C. Methanol (10 ml) was added and the mixture wasstirred for additional 20 min. at room temperature, evaporated andpartitioned between ethyl acetate and water. The organic phase waswashed subsequently with water, saturated aqueous NaHCO₃, water,saturated aqueous NaCl, dried (Na₂SO₄), filtered and evaporated.Chromatography (SiO₂, ethyl acetate-heptane 1:4) gave the benzoate (1.64g, 87%).

[α]_(D) ²²: +0.95° (c 1.0, chloroform)

¹H NMR data (CDCl₃) δ: 8.04-7.98 (m, 2H), 7.56 (tt, 1H, J=7.5 and 1.3Hz), 7.43 (t, further coupled, 2H, J=7.5 Hz), 7.26 and 6.83 (AB pattern,further coupled, 4H, J=8.6 Hz), 4.62 and 4.50 (AB pattern, 2H, J=11.3Hz, ArCH ₂), 4.63 (d, further coupled, J=1.3 Hz, H-1), 4.54 (A part ofan ABX system, 1H, J=11.8 and 2.7 Hz, H-6_(a)), 4.47 (B part of an ABXsystem, 1H, J=11.8 and 5.5 Hz, H-6_(b)), 4.27 (ddd, 1H, J=8.4, 5.5 and2.7 Hz, H-5), 3.99-3.92 (2H, H-3 and H-4), 3.74 (s, 3H, CH₃OAr),3.67-3.48 (3H, CH ₂CH₃ and H-2) and 1.22 (t, 3H, J=7.0 Hz, CH₂CH ₃) ppm.

¹³C NMR data (CDCl₃) δ: 166.2, 159.5, 132.9, 130.1, 129.9, 129.6, 129.2,128.3, 113.9, 99.6, 76.72, 72.0, 71.7, 62.3, 62.2, 64.1, 58.5, 55.4,55.2 and 15.4 ppm.

Methyl6-O-benzoyl-3-deoxy-2-O-ethyl-4-O-p-methoxybenzyl-3-sulfamino-α-D-altropyranosideSodium Salt (Cf. H. P. Wessel; J. Carbohydr. Chem. 11 (8), (1992),1039-1052).

To a stirred solution of methyl3-azido-6-O-benzoyl-3-deoxy-2-O-ethyl-4-O-p-methoxybenzyl-α-D-altropyranoside(620 mg, 1.31 mmol) in tetrahydrofuran (40 ml) was added water (265 ml)and triphenylphosphine (1.7 g, 6.5 mmol). The solution was stirred for24 h and was then concentrated to 12 ml. The residue was diluted withmethanol (10 ml) and water (3 ml). To the stirred solution was thenadded trimethylamine sulfurtrioxide complex (370 mg, 2.7 mmol). The pHwas adjusted to 8.5 by 1 M aqueous NaOH. After a few minutes aprecipitate was formed, which was dissolved by adding tetrahydrofuran (3ml) and methanol (4 ml). The reaction mixture was stirred for 0.5 h,after which time additional trimethylamine sulfurtrioxide complex (86mg) was added. The stirring was continued for 15 min. During the courseof the reaction pH was kept at 8-9 by adding 1 M aqueous NaOH. Themixture was then diluted with water (7 ml) and the organic solvents wereevaporated under reduced pressure. Additional water (5 ml) was added andthe suspension was lyophilised and chromatographed (SiO₂,chloroform-methanol-water 100:15:1→70:30:5) to give the sulfaminocompound, presumably in the sodium salt form, (668 mg, 92%) afterlyophilisation from water. A sample was crystallized from aqueousacetone.

m.p. 112-115° C. (aqueous acetone).

[α]_(D) ²²: +156.4° (c0.5, water)

¹H NMR data (CD₃OD) δ: 7.94-7.89 (m, 2H), 2H, 7.64-7.56 (m. 1H), 7.44(m, 2H), 7.28 and 6.73 (AB pattern, further coupled, 4H, J=8.6 Hz), 4.71and 4.49 (AB pattern, 2H, J=11.4 Hz, ArCH ₂), 4.68 (bs, 1H, H-1), 4.51(dd, 1H, J=11.6 and 2.4 Hz, H-6_(a)), 4.44 (dd, 1H, J=11.6 and 4.4 Hz,H-6_(b)), 4.09 (unresolved ddd, 1H, J≈4, 3 and 1 Hz, H-3), 4.00-3.92(2H, H-5 and H-2), 3.86 (dd, 1H, J=9.9 and 4.2 Hz, H-4), 3.71-3.54 (5H,CH ₂CH₃ and CH₃OAr), 3.65 (s, CH₃OAr), 3.40 (s, 3H, CH₃O) and 1.17 (t,3H, J=7.0 Hz, CH₂CH ₃) ppm.

¹³C NMR data (CD₃OD) δ: 167.8, 160.8, 134.2, 131.4, 130.9, 130.6, 129.5,114.7, 101.6, 77.03, 70.02, 69.2, 67.0, 66.4, 65.2, 55.6, 55.5, 50.6 and15.8 ppm.

Methyl 6-O-benzoyl-3-deoxy-2-O-ethyl-3-sulfamino-α-D-altropyranosideAmmonium Salt (bpy_(—)30)

Methyl6-O-benzoyl-3-deoxy-2O-ethyl-4-O-p-methoxybenzyl-3-sulfamino-α-D-altro-pyranosidesodium salt (334 mg, 2.0 mmol) was hydrogenolysed for 6 h in glacialacetic acid (55 ml) at 0.23 MPa and room temperature, using 5% palladiumon charcoal as a catalyst. Filtering and lyophilisation gave a residuewhich was chromatographed (SiO₂, chloroform-methanol-water100:15:1→70:30:5). The product was dissolved in water and passed througha cation exchange resin (BIO-REX® 70, 200-400 mesh, ammonium form) andlyophilised to give the title compound (222 mg, 86%)

[α]_(D) ²²: +55.70° (c 1.3, water)

¹H NMR data (pyridine-d₅) δ: 8.23-8.17 (2H), 7.52-7.45 (1H), 7.42-7.35(2H), 5.09 (dd, 1H, J=11.65 and 1.54 Hz, H-6_(a)), 4.82 (dd, 1H, J=11.43and 6.81 Hz, H-6_(b)), 4.82 (s, 1H, H-1), 4.66 (unresolved dd, 1H, J≈3.5and 3.5 Hz, H-3), 4.36 (dd, 1H, J=10.11 and 3.96 Hz, H-4), 4.32-4-23(1H, H-5), 4.18 (d, 1H, J=3.08 Hz, H-2), 3.60-3.44 (2H, CH ₂CH₃), 3.16(s, 3H, CH₃O) and 1.00 (t, 3H, J=7.0 Hz, CH₂CH ₃) ppm.

¹H NMR data (D₂O, ref. acetone at 2.35 ppm) δ: 8.22-8.17 (2H), 7.83 (tt,1H, J=7.5 and <2 Hz), 7.72-7.65 (2H), 4.97 (HDO), 4.93 (s, 1H, H-1),4.81 (dd, virtually coupled, 1H, J=12 and 1.7 Hz, H-6_(a)), 4.65 (dd,virtually coupled, 1H, J=12 and 5.5 Hz, H-⁶b), 4.18-4-15 (2H, h-4 andH-5), 4.05 (dd, 1 H. J=3.3 and 1.4 Hz, H-2), 3.95-3.75 (sym. m. 2H, CH₂CH₃), 3.94-3.91 (m, 1H, H-3), 3.54 (s, 3H, CH₃O) and 1.35 (t, 3H, J=7Hz, CH₂CH ₃) ppm (the assignments of the ¹H NMR spectrum signals weremade on the basis of a COSY experiment).

¹³C NMR data (D₂O, ref.: acetone at 33.19 ppm) δ171.4, 136.9, 132.5,132.1, 131.7, 102.5, 78.4, 69.9, 69.1, 67.4, 65.7, 58.1, 56.0 and 17.5ppm.

Methyl3-azido-6-O-pivaloyl-3-deoxy-2-O-ethyl-4-O-p-methoxybenzyl-α-D-altropyranoside

A solution of methyl3-azido-3-deoxy-2-O-ethyl-4-O-p-methoxybenzyl-α-D-altro-pyranoside (476mg, 1.3 mmol) in pyridine (8 ml) was stirred at 0° C. Pivaloyl chloride(390 μl, 3.25 mmol) was added and the temperature was raised to roomtemperature. The stirring was continued for 1.5 h and the mixture wasthen cooled to 0° C. Methanol (10 ml) was added and the mixture wasstirred for additional 20 min. at room temperature, evaporated andpartitioned between ethyl acetate and water. The organic phase waswashed subsequently with water, saturated aqueous NaHCO₃, water,saturated aqueous NaCl, dried (Na₂SO₄), filtered and evaporated.Chromatography (SiO₂, methyl tert-butyl ether-heptane 1:4) and (C-18Lobar, Merck, acetonitrile-water 4:1) gave the pivaloate (431 mg, 75%).

¹H NMR data (CDCl₃) δ: 7.28 and 6.89 (AB pattern, further coupled, 4H,J=8.6 Hz), 4.60 and 4.49 (AB pattern, 2H, J=11.0 Hz, ArCH ₂), 4.57 (s,1H, H-1), 4.39 (dd, 1H, J=14.5 and 5.3 Hz, H-6_(a)), 4.13 (2H, H-6_(b)and H-4), 3.93 (m, 1H, H-3), 3.84-3.78 (4H, H-5 and MeOAr), 3.64-3.45(3H, CH ₂CH₃ and H-2), 3.39 (s, 3H, 1-OMe) and 1.22-1.15 (s and t, 12H,J=7.0 Hz, C(CH₃)₃ and CH₂CH ₃) ppm.

¹³C NMR data (CDCl₃) δ: 178.1, 159.6, 129.9, 129.3, 113.9, 99.5 (C-1),76.7 (C-2), 72.5 (C-5), 71.9 (ArCH₂), 66.4 (C-4), 66.2 (CH₂CH₃), 63.6(C-6), 58.5, 55.3, 55.2, 38.8, 27.2 and 15.4 ppm.

Methyl6-O-pivaloyl-3-deoxy-2-O-ethyl-4-O-p-methoxybenzyl-3-sulfamino-α-D-altropyranosideSodium Salt

(Cf. H. P. Wessel; J. Carbohydr. Chem. 11 (8), (1992), 1039-1052.)

To a stirred solution of methyl3-azido-6-O-pivaloyl-3-deoxy-2-O-ethyl-4-O-p-methoxybenzyl-α-D-altropyranoside(300 mg, 0.683 mmol) in tetrahydrofuran (20 ml) was added water (150 ml)and triphenylphosphine (0.90 g, 3.3 mmol). The solution was stirred for24 h and was then concentrated to 5 ml. The residue was diluted withmethanol (5 ml) and water (1 ml). One half of this solution was used toprepare the corresponding oxamate (see 48.182). To the remaining half ofthe stirred solution (0.342 mmol) was then added trimethylaminesulfurtrioxide complex (99 mg, 0.719 mmol). The pH was. adjusted to 8.5by 1M aqueous NaOH. After a few minutes a precipitate.was formed, whichwas dissolved by adding tetrahydrofuran (0.7 ml) and methanol (1 ml).The reaction mixture was stirred for 0.5 h, after which time additionaltrimethylamine sulfurtrioxide complex (45 mg) was added The stirring wascontinued for 15 min. During the course of the reaction pH was kept at8-9 by adding 1M aqueous NaOH. The mixture was then diluted with water(2 ml) and the organic solvents were evaporated under reduced pressure.Additional water (5 ml) was added and the suspension was lyophilised andchromatographed (SiO₂, chloroform-methanol-water 100:15:1→80:20:1) togive the sulfamino compound, presumably in the sodium salt form, (161mg, 80%) after lyophilisation from water.

¹H NMR data (dmso-D₆) δ: 7.24 and 6.87 (AB pattern, further coupled, 4H,J=8.8 Hz), 4.69 and 4.24 (AB pattern, 2H, J=10.8 Hz, ArCH ₂), 4.58 (s,1H, H-1), 4.29 (dd, 1H, J=11.4 and 1.7 Hz, H-6_(a)), 4.05-3.96 (2H,H-6_(b) and NH), 3.83-3.76 (m, 2 H, H-2 and H-3), 3.76-3.73 (4H, s andm, H-5 and MeOAr), 3.54-3.40 (3H, CH ₂CH₃ and H-4), 3.27 (s, 3H, 1-OMe)and 1.12-1.02 (s and t, 12H, J=7.0 Hz, C(CH₃)₃ and CH₂CH ₃) ppm.

Methyl 6-O-pivaloyl-3-deoxy-2-O-ethyl-3-sulfamino-α-D-altropyranosideAmmonium Salt (bpy_(—)37)

Methyl6-O-pivaloyl-3-deoxy-2-O-ethyl-4-O-p-methoxybenzyl-3-sulfamino-α-D-altro-pyranosidesodium salt (153 mg, 0.259 mmol) was hydrogenolysed for 6 h in glacialacetic acid (8 ml) at 0.23 MPa and room temperature, using 5% palladiumon charcoal as a catalyst. Filtering and lyophilisation gave a residuewhich was chromatographed (SiO₂, chloroform-methanol-water 80:20:1 with0.1% NH₃) to give the title compound (77.2 mg, 74%).

¹H NMR data (D₂O, ref. acetone at 2.35 ppm) δ: 4.83 (HDO), 4.78 (s 1H,H-1), 4.43 (dd, virtually coupled, 1H, J=11.5 and 1.1 Hz, H-6_(a)), 4.30(dd, virtually coupled, 1H, J=11.5 and 5.1 Hz, H-6_(b)), 3.95-3.91 (2H,H-4 and H-5), 3.89 (dd, 1H, J=3.3 and 1.3 Hz, H-2), 3.80-3.62 (m. 3H,H-3 and CH ₂CH₃), 3.42 (s, 3H, CH₃O) and 1.24-1.21 (s znd t, 12H, J=7Hz, ^(t)Bu and CH₂CH ₃) ppm (the assignments of the ¹H NMR spectrumsignals were made on the basis of a COSY experiment).

³C NMR data (D₂O, ref.: acetone at 33.19 ppm) δ188.3, 106.2, 82.2, 73.8,72.8, 70.6, 69.3, 61.9, 59.8,45.6, 37.1 and 21.3 ppm.

Methyl6-O-pivaloyl-3-deoxy-2-O-ethyl-4-O-p-imethoxybenzyl-3-^(t)butyloxamido-α-D-altropyranoside

Oxalyl chloride (120 μl, 1.37 mmol) dissolved in tetrahydrofurane (1 ml)was cooled to −26° C. A solution of t-butanol (131 mg, 1.77 mmol) andpyridine (154 μl, 1.91 mmol) in tetrahydrofurane (2 ml) was addeddropwise and the mixture was stirred at −26° C. for 15 min, while theinitially formed yellowish precipitate turned white.

The solvents were removed under reduced pressure from thetetrahydrofurane solution from the reduction of3-azido-6-O-pivaloyl-3-deoxy-2-O-ethyl-4-O-p-methoxybenzyl-α-D-altropyranoside(150 mg, 0.342 mmol). The residue was redissolved in tetrahydrofurane(2.5 ml) and pyridine (154 μl) and added dropwise to the cooled solutionabove and stirred at −26° C. for 1 h. Water (2 ml) was added and themixture diluted with t-butyl methyl ether (15 ml) and washed with water,saturated sodium hydrogen carbonate and brine. The organic solution wasdried over sodium sulfate and the solvents were removed under reducedpressure . The residue was chromatographed (SiO₂, t-butyl methylether-toluene 1:8) to yield 187 mg, 100% of the title compound.

¹H NMR data (CDCl₃) δ: 8.25 (d, 1H, 10.1 Hz, 3-NH), 7.26 and 6.85 (ABpattern, further coupled, 4H, J=t8.6 Hz), 4.82-4.71 (m and d, 2H, 10.5Hz, H-3 and ArCH ₂), 4.69 (s, 1H, H-1), 4.42 (dd, 1H, 1.7 and 11.7 Hz,H-b 6 _(a)) 4.31 (d, 1H, 10.5 Hz, ArCH ₂), 4.16 (dd, 1H, 6.2 and 11.7Hz, H-6_(b)), 3.88 (ddd, 1H, 1.8, 5.9 and 10.3 Hz, H-5), 3.81-3.75 (ddand s, 4H, H-4 and ArOMe), 3.64-3.49 (m, 2H, CH ₂CH₃), 3.47-3.43 (dd ands, 4H, 1.3 and 3.1 Hz, H-2 and 1-OMe), 1.55 (s, 9H, oxamate C(CH₃)₃),1.19 and 1.18 (s and t, 12H, 7 Hz, 6-O pivaloate ^(t)Bu and CH₂CH ₃)ppm.

¹³C NMR data (CDCl₃) δ: 178.1, 159.4, 159.1, 157.4, 130.3, 129.4, 113.8,98.9, 84.2, 77.2, 75.9, 70.8, 69.5, 65.8, 65.7, 63.4, 55.2, 55.1, 45.7,38.8, 27.7, 27.1, 15.3 ppm.

Methyl 6-O-pivaloyl-3-deoxy-2-O-ethyl-3-oxamido-α-D-altropyranosideAmmonium Salt (bpy_(—)54)

Methyl6-O-pivaloyl-3-deoxy-2-O-echyl-4-O-p-methoxybenzyl-3-^(t)butyloxamido-α-D-altropyranoside(180 mg, 332 μmol) was dissolved in acetic acid-ethanol mixture (1:1, 6ml), palladium on carbon (5%, 250 mg) was added and subjected forhydrogenolysis at 39 psi overnight. The catalyst was filtered of on acelite pad, washed with ethanol. The filtrate was concentrated underreduced pressure, redissolved in dichloromethane (2.4 ml).Trifruoroacetic acid (960 μl) was added and the mixture stirred for 4.5h at room temperature. Water (3 ml) and ammonia (3M, 300 μl) was addedand reaction mixture was concentrated under reduced pressure andlyophilized. The residue was chromatographed (SiO₂,chloroform-methanol-water 80:20:1 with 0.1% NH₃) to give the titlecompound, 117.1 mg, 89%.

¹H NMR data (D₂O, ref.: acetone at 2.35 ppm) δ: 4.80 (HDO), 4.78 (s 1H,H-1), 4.45-4.35 (m, 2H, H-3 and H-6_(a)), 4.26 (dd, 1H, 4.4 and 12.1 Hz,H-6_(b)), 4.07-3.96 (m, 2H, H-4 and H-5), 3.77-3.58 (m, 3H, CH ₂CH₃ andH-2), 3.45 (s, 3H, 1-OMe), 1.20-1.13 (s and t, 12H, 6-O-pivaloate ^(t)Buand CH₂CH ₃) ppm.

¹³C NMR data (D₂O, ref.: acetone at 33.19 ppm) δ: 188.1, 171.9, 171.4,105.7, 82.4, 73.7, 72.9, 70.3, 69.7, 62.0, 56.1, 45.9, 37.0, 21.2 ppm.

Methyl3-azido-6-O-pyrrol-3′-ylcarboxyl-3-deoxy-2-O-ethyl-4-O-p-methoxybenzyl-α-D-altropyranoside

A solution of methyl3-azido-3-deoxy-2-O-ethyl-4-O-p-methoxybenzyl-α-D-altro-pyranoside (476mg, 1.3 mmol) in pyridine (496 μl) and dichloromethane (2 ml) wasstirred at 0° C. Methanesulfonyl chloride (380 μl, 4.90 mmol) was addedand the temperature was raised to room temperature. The stirring wascontinued for 5 h and the mixture was then cooled to 0° C. Water (1 ml)was added and the mixture was stirred for additional 20 min. at roomtemperature, evaporated and partitioned between ethyl acetate and water.The organic phase was washed subsequently with water, saturated aqueousNaHCO₃, water, saturated aqueous NaCl, dried (Na₂SO₄), filtered andevaporated. Chromatography (SiO₂, methyl tert-butyl ether-toluene 1:9)yielded 216 mg (79%) of the mesylate as a yellowish oil.N-Triisopropylsilyl pyrrole-3-carboxylic acid (484 mg, 1.81 mmol) and1,8-diazacicyclo[5.4.0]undec-7-ene (255 μl) were dissolved indimethylformamide (1 ml) and added to a solution of the mesylate abovein dimethylformamide (1 ml). The mixture was heated at 90° C. overnightand the subjected to chromatography (210 g C-8 Lobar, Merck,acetonitrile-water 3:2) to yield 30 mg, 16% of the pyrroloyl ester.

¹H NMR data (CDCl₃) δ: 8.73 (br.s., 1H, pyrrole NH), 7.39 (symm. m., 1H,pyrrole H-4), 7.27 and 6.85 (AB pattern, further coupled, 4H, J=8.8 Hz),3.74 (symm. m, 1H, pyrrole H-2), 3.64 (symm. m, 1H, pyrrole H-5),4.63-4.49 (s and 2 d, 3H, H-1 and ArCH ₂), 4.48-4.36 (m, 2H, 2 H-6),4.22 (symm. m, 1H, H-5), 3.94-3.84 (m, 2H, H-3 and H-4), 3.78 (s, 3H,ArOMe), 3.68-3.55 (m, 3H, CH ₂CH₃ and H-2), 3.40 (s, 3H, 1-OMe), 1.20(t, 3H, 6.8 Hz, CH₂CH ₃) ppm.

¹³C NMR data (CDCl₃) δ: 164.6, 159.5, 129.9, 129.4, 123.6, 11.7, 116.1,113.9, 109.9, 99.9, 76.9, 72.5, 72.0, 66.8, 66.3, 63.0, 58.9, 55.3,55.2, 15.4 ppm.

Methyl6-O-pyrrol-3′-ylcarboxyl-3-deoxy-2-O-ethyl-3-sulfamino-α-D-altropyranosideAmmonium Salt (bpy_(—)40)

To a stirred solution of methyl3-azido-6-O-pivaloyl-3-deoxy-2-O-ethyl-4-O-p-methoxybenzyl-α-D-altropyranoside(30 mg, 61 μmol) in tetrahydrofuran (1 ml) was added water (11 μl) andtriphenylphosphine (83 mg, 305 μmol). The solution was stirred for 24 h,diluted with and water (1 ml). Trimethylamine sulfurtrioxide complex (9mg, 0.72 μmol) was then added. The pH was adjusted to 8.5 by 1 M aqueousNaOH. After a few minutes a precipitate was formed, which was dissolvedby adding tetrahydrofuran (0.7 ml) and methanol (1 ml). The reactionmixture was stirred for 0.5 h, after which time additionaltrimethylamine sulfurtrioxide complex (9 mg) was added. The stirring wascontinued for 15 min. During the course of the reaction pH was kept at8-9 by adding 1M aqueous NaOH. The mixture was then diluted with water(2 ml), washed with t-butyl methyl ether, lyophilised andchromatographed (SiO₂, chloroform-methanol-water 80:20:1) to give thesulfamino compound, presumably in the sodium salt form, (30.6 mg, 93%)after lyophilisation from water.

¹H NMR data (CD₃OD) δ: 7.35 (unresol. dd, x part of a ABX-pattern, 1H,J_(AX)+J_(BX)=3.5 Hz, pyrrole H-2), 7.29 and 6.79 (AB pattern, furthercoupled, 4H, J=8.6 Hz), 6.75 (dd, 1H, 2.0 and 2.9 Hz, pyrrole H-4), 6.51(dd, 1H, 1.3 and 2.9 Hz, pyrrole H-5), 4.71 and 4.46 (AB pattern, 2H,J=11.4 Hz, ArCH ₂), 4.66 (bs, 1H, H-1), 4.39 and 4.33 (2 dd, 2H, 2.6,5.1 and 11.7 Hz, 2 H-6), 4.04 (m, 1H, H-3), 3.97-3.87 (m and dd, 2H, 1.1and 3.1 Hz, H-5 and H-2), 3.80 (dd, 1H, 4.4 and 10.1 Hz, H-4) 3.73 (s,3H, ArOMe), 3.68-3.52 (m, 2H, CH ₂CH₃), 3.39 (s, 3H, 1-OMe), 1.15 (t,3H, 7.0 Hz, CH₂CH ₃) ppm.

¹³C NMR data (CD₃OD) δ: 167.32, 160.9, 131.3, 131.0 125.3, 120.1, 116.3,114.7, 110.2, 101.7, 77.2, 10.4, 69.9, 67.2, 66.4, 55.6, 55.5, 50.9 and15.8 ppm.

The residue was dissolved in methanol (15 ml), palladium on carbon (5%,50 mg) was added and the mixture hydrogenolyzed at 30 psi for 3 h. Thecatalyst was filtered off, the filtrate concentrated under reducedpressure, redissolved in ammonia (2%) in methanol-water (1:1, 0.6 ml)and chromatographed (3 ml C-18 Supelco, methanol-water 2:3). Methanolwas removed under reduced pressure from the pooled product-containingfractions and the residue lyophilized to give 6.2 mg of the titlecompound as the ammonium salt.

¹H NMR data (CD₃OD) δ: 7.35 (m, 1H, pyrrole H-2), 6.75 (dd, 1H, 2.0 and2.9 Hz, pyrrole H-4), 6.55 (dd, 1H, 1.3 and 2.9 Hz, pyrrole H-5), 4.67(br.s, 1H, H1), 4.55 (dd, 1H, 1.3 and 11.4 Hz, H-6_(a)), 4.31 (dd, 1H,5.9 and 11.6 Hz, H-6_(b)), 3.86-3.76 (m, 4H, H-2, H-3, H-4, H-5),3.75-3.56 (m, 2H, CH ₂CH₃), 3.40 (s, 3H, 1-OMe), 1.21 (t, 3H, Hz,CH₂CH₃) ppm.

Example 6

Chaperone Assays and the Use Thereof in Testing Compounds For Inhibitionof Chaperone Binding

In order to conclusively identify peptides or peptide mimetics that bindstrongly to PapD, a good assay is required. The maltose binding protein(MBP) has been engineered by the inventors so that it is recognized byPapD. The commercially available plasmid pMAL-P2 encoding MBP with alinker arm encoding sequence fused to the 3′ end of the gene was used.MBP is secreted into the periplasmic space and is easily purified byaffinity chromatography using amylose resin. MBP is eluted from thecolumn with 20 mM maltose. PapD is not co-eluted with MBP whenco-expressed with ISP whereas fusing the PapG COOH-terminus to MBPresults in PapD binding to MBP to form a complex that is co-eluted fromthe affinity column. Using this strategy, it has been found that fusingthe carboxy terminal 140 (MBP-G1′-140′) and 134 (MBP-G134′) amino acidsof PapG to MBP results in the formation of a strong PapD-MBP complexwhich is purified by maltose affinity chromatography. The complexbetween the MBP fusion and PapD has been shown to be stable in up to 3 Murea similar to the stability of a PapD-PapG complex arguing that theCOOH-terminal 134 amino acids of PapG probably contains most of thechaperone recognition motif. In addition, binding to the MBP fusionprotein has proven dependent on the universal chaperone anchoringresidues, Arg-8 and Lys-112, as described herein since mutations inthese two residues abolished PapD binding to the MBP fusion protein.

It is also interesting to test the ability of MBP-G fusions to inhibitpilus assembly and thus bacterial attachment by co-expressing the fusionproteins in cells producing P pili. If PapD binds to the MBP-G fusion,it will be titrated away from pilus subunits and thus decrease orabolish pilus formation. This technique will be one mechanism tovalidate the concept that chaperone binding peptides can abolish thevirulence of a pathogen by preventing the assembly of surface-localizedadhesins. Using this analysis it has been found that in strainsproducing P pili, co-expression of the MBP-G1′-140′ and MBP-G134′ fusioncompletely abolishes the ability of the bacteria to bind red blood cellsand cause hemagglutionation. Further, it has recently been shown by theinventors that after coexpression of the MBP-G1′-140′ fusion inHB101/pPAP5-bacteria, it was not possible to detect any visual signs ofpili on the surface of the bacteria by electron microscopy. This isevidence for the theory expressed in this invention, that theprevention/inhibition of binding between pilus subunits and molecularchaperones also prevents the assembly of intact pili.

These results support the biological relevance of the crystal structuredescribed in FIG. 3, showing that other proteins can be engineered to berecognized by PapD by fusing the carboxy terminal recognition site ontothat protein. The MBP-G134′ and MBP-G1′-140′ fusions have been purifiedto establish in vitro assays to measure interactions with PapD. Thepurified fusion proteins are coated on wells of microtiter plates andthe ability of PapD to bind to the fusion proteins is tested in an ELISAprocedure. This assay is critical since it allows testing the ability ofcompounds to inhibit binding of PapD to the domain on PapG recognized bythe chaperone. Furthermore, the purified fusion proteins can be used inthe Pharmacia BiaCore® assay to quantitate binding to PapD and establishinhibition assays for compounds of the invention. Carboxyl-terminalpeptides have already been suggested to correspond to part of thechaperone recognition motif. One can thus investigate whether thecarboxyl terminal peptides are able to inhibit binding of PapD to theMBP-G1′-140′ fusion in the ELISA assay. The development of this highthrough-put assay makes it possible to screen peptide libraries,chemical libraries, natural compounds, and peptide mimetics for theirability to inhibit chaperone binding. In addition, this assay can beused to test whether the known periplasmic chaperones utilize commonrecognition paradigms. In addition to testing compounds for theirability to inhibit binding to the MBP-G fusion, other assays have beenestablished which measure PapD-peptide interactions. For example, anELISA has been developed to measure the binding of PapD to peptidescoated on wells of microtiter plates. In this assay the octamericpeptide G1′-8′WT was an equally efficient inhibitor as the 19-merG1′-19′WT, revealing the octamer to be the optimal starting point fordesigning of modified compounds with increased affinity for the bindingsite of PapD (cf. Example 7).

Also, as previously described, native PapD is able to bind to reduced,denatured PapG and restore the PapD-PapG complex in vitro in areconstitution assay (Kuehn et al., 1991). This assay has been proposedto reflect the recognition function of PapD in vivo and will be used todetermine the ability of the compounds to inhibit PapD binding to PapGin vitro. For example, the carboxyl terminal PapG peptide has been shownto inhibit the binding of PapD to PapG in this assay, establishing thatit occupies the pilus subunit binding site of PapD. Another way to mapPapD-peptide interactions is to test the ability of compounds to reducethe rate of well defined proteolytic cleavage events. For example,partial digestion with trypsin cleaves PapD in the P1-G1 loop at residueLys-99 (See “T” site denoted in FIG. 3). The rate of tryptic cleavage ofPapD was reduced by preincubation of PapD with PapG and PapK peptides(cf. Example 2). The observed protection of PapD by bound peptides maybe due to a change of the local conformation of the F1-G1 loop, or dueto physical contact of the loop by the peptide. These assays may thus beused as an initial screening for the ability of new compounds to bind toPapD and interfere with its recognition function. Strong bindingcompounds that inhibit PapD binding to the MBP-G1′-140′ fusion will beco-crystallized with PapD to provide the structural basis of therecognition surface used by PapD. As new crystallographic data becomesavailable, the relevance of critical PapD-inhibitor or PapD-enhancerinteractions will be tested by determining the effect of site-directedmutations on the ability of the chaperone to bind pilus subunits andmediate pilus assembly. This important information will lead to defininghow chaperones recognize subunits and the function of chaperone-subunitinteractions.

A quantitative chaperone binding assay has also been developed: Amodified PapG peptide has been synthesized, wherein Ser-9′ issubstituted by a Cys. From the crystal structure of PapD bound to thepeptide, the side chain of Ser-9′ was not predicted to interact withPapD. Instead, this side chain was oriented towards the solvent, butSer-9′ was adjacent to the last amino acid of the interactive zipperbetween PapD and the peptide. The environmentally sensitive fluorescentprobe 5-IAF (5-iodoacetamidfluorescein) was then covalently coupled tothe peptide via the sulfhydryl group on the Cys residue and the labelledpeptide was purified; of course any other suitable envirorenmentallysensitive fluorescent probe may be employed. It was found that theaddition of PapD to the peptide causes a marked decrease in thefluorescent intensity and a shift in the emission maxima. Thisinformation was used to calculate a binding constant for thePapD-peptide interaction. The change in the fluorescence intensity at514 nm after the addition of increasing concentrations of PapD wasdetermined. By plotting the fluorescent intensities versus PapDconcentrations, a binding constant of 2.5×10⁻⁶ M was calculated (Thecalculations were performed on the commercially available computerprogram “Kaleidagraph”).

It is thus possible to evaluate the binding constants of othersubstances binding to PapD in a quantitative manner, as the addition ofa substance which binds competitively to PapD will result in anincreased fluorescence compared to a situation where less or nocompetitive substance is present in the system.

A similar assay system will be developed using MBP-G1′-140′ instead ofthe PapG peptide as described herein. As this fusion protein has beenfound to interact with two binding sites in PapD, it will be possibleto 1) quantitate the binding affinity to the second binding site (ondomain 2) and 2) screen for compounds which interact with either of thetwo binding sites and quantitate the interaction. Of course, labellingthe site 2 peptide with a suitable environmentally sensitive probeshould allow a determination of the binding constant using the samemethodology as describe above.

The discovery that the DegP protease is greatly responsible for thedegradation of pilin subunits in the absence of a chaperone renders thedegP41 strain an interesting candidate for an in vivo test system of theeffects exerted on PapD by compounds of the invention.

When administering a substance which prevents, inhibits or enhances thebinding between the chaperone and the pilus subunits to a systemcontaining the degP41 strain, the substance should, even in smallamounts, be toxic to the bacteria, as the DegP⁻ bacteria will beincapable of degrading the accumulating pilus subunits. Note that adegP41 strain was used to identify a hitherto unknown binding site ondomain 2 of PapD, cf. example 10.

However, such an assay requires that the tested substance is able toenter the periplasmic space before it can exert its effect; this factrenders this type of assay less suitable as a screening assay for leadcompounds, as it will “ignore” substances which have the desired effectson chaperone-subunit interaction, but which are e.g. too hydrophillic toenter the periplasmic space. On the other hand, the system will bewell-suited for assessing the clinical potential of substances whichhave already proven successful in the in vitro assays described herein.

Once the model PapD system is in place, the experiments described abovecan of course be expanded to include the other members of the PapD-likefamily of chaperones. In this way it will be possible to establish thegeneral requirements for chaperone recognition and the molecular basisof the chaperone recognition paradigm in gram negative bacteria.

Some specific assays for determination of interactions with molecularchaperones are described in detail in Example 10.

Example 7

Design of Peptides and Peptide Mimetics Capable of Binding to theBinding Site of PapD and Other Pilus Assembling Chaperones

To provide further information on chaperone-subunit interactions, it iscontemplated to synthesize peptides overlapping the entire PapG proteinand other pilus subunits. In addition, probing peptide binding toavailable chaperones using both a chemical peptide library and a phagedisplay library should be performed. The two libraries are complementarysince the peptides are presented in different environments which couldimportantly influence binding to the chaperones. In practice thechemical library is limited to all possible hexapeptides and to hepta-and octapeptides if some of the natural amino acids are excluded or ifone or two residues are kept invariant. This library can be evaluated bydirect binding of the chaperone to the resin beads and sequencing ofinteresting peptides. The phage display library contains approximately10¹⁰ peptides in the PIII protein of the so-called “fusion phage” thatretains phage function and displays the foreign peptides on the surface.The ability of peptide-containing phages to bind PapD coated in wells ofmicrotiter plates can be detected in an ELISA using the techniquesdescribed in Example 6. Positive binding phages will be amplified,purified and retested for their ability to bind PapD. The sequence ofthe peptide insert of positively binding phages will be determined andthe corresponding peptides will be synthesized and tested for theirability to inhibit biding of PapD to the MBP-G fusion as described inExample 6.

The results from the studies outlined above will be of the utmostimportance for design and evaluation of ligands acting as “chaperoneinhibitors”. In spite of this, the crystal structure of the PapD-PapG19-mer peptide complex provides sufficient insight into chaperone-ligandinteractions to initiate systematic studies of small peptides andpeptide mimetics as chaperone inhibitors already at the present stage.The high inhibitory power of the PapG 8-mer peptide to PapD (equal tothe PapG 19-mer peptide, cf. Example 6) reveals the feasibility of suchstudies and the conserved features of the proposed binding site on thechaperones indicate that such inhibitors could well have broadspecificity.

The specificity in recognition of the PapG 19-mer by PapD has beensuggested to be provided by anchoring of the peptide's carboxyl terminusto the PapD cleft residues Arg-8 and Lys-112 and subsequent “zippering”interactions between alternating hydrophobic residues in the peptide andcomplementary hydrophobic residues in PapD. The following experimentssupport this hypothesis. Firstly, mutations in the anchor residues Arg-8and Lys-112 have abolished subunit binding in vivo. Secondly, deletionof the C-terminal residue in the PapG 19-mer leads to a substantiallydecreased binding to PapD as the hydrophobic “zippering” interactionsbetween peptide and PapD were then placed off register when theC-terminus of the deletion peptide was anchored to Arg-8 and Lys-112.The “zippering” hypothesis for specificity in binding between PapD andpeptides (or pilus subunits) has been preliminary investigated byperforming the following experiments:

A length series of peptides form the C-terminus of PapG consisting ofG1′-5′, G1′-6′, G1′-7′, G1′-8′, G1′-11′, G1′-16′ and G1′-19′ wasprepared. A replacement series and a deletion series of G1′-8′ was alsosynthesized. In the replacement series Pro-1′, Phe-2′, Leu-4′, Val-5′,Met-6′ and Met-8′ was replaced by Ser, whereas Ser-3′ and Thr-7′ wasreplaced by Ala (i.e. hydrophobic amino acids was replaced by the polarSer and hydrophillic amino acids was replaced by Ala). In the deletionseries one residue at a time in G1′-8′ was deleted with simultaneousaddition of a serine (which is found in position 9′ in native PapG) tomaintain the peptide length at 8 amino acids. The peptides were allsynthesized by the Fmoc solid phase strategy and were purified byreversed phase HPLC.

The ability of the peptides to inhibit the binding of PapD to theMBP-G1′-140′ fusion protein was then investigated using the followingELISA test:

Stock solutions of MBP-G1′-140′ proteins in PBS were diluted to 0.1 μMwith PBS. The wells of microtiter plates were coated with 50 μl of theprotein solutions overnight at 4° C. The wells were washed with PBS, andblocked with 200 μl of 3% bovine serum albumin (BSA) in PBS for 2 hoursat 25° C. The plates were washed vigorously three times with PBS andincubated with 50 μl of 1-5 μM PapD proteins in 3% BSA-PBS for 45minutes at 25° C. The PapD was preincubated with each of the peptides ata 1:25 ratio for 30 min before being added to the wells. After threewashings with PBS, the wells were incubated with a 1:500 dilution ofrabbit anti-PapD antiserum in 3% BSA-PBS for 45 minutes at 25° C. Afterthree washings with PBS, the wells were incubated with a 1:1000 dilutionof goat antiserum to rabbit IgG coupled to alkaline phosphatase in 3%BSA-PBS for 45 minutes at 25° C. After three washings with PBS and threewashings with developing buffer (10 mM diethanolamine, 0.5 mM MgCl₂), 50μl filtered 1 mg/ml p-nitrophenyl phosphate in developing buffer wasadded, the reaction was incubated for 1 hour or longer if necessary inthe dark at 25° C., and the absorbance at 405 nm was read.

The inhibitory powers of the peptides in the three series are presentedin FIGS. 17-19 and the number of experiments performed with each seriesis given in the FIGS. Vertical lines for each peptide in the figures are95% confidence intervals obtained after a statistical analysis of theexperimental data. As revealed by the evaluation of the length series,the peptides G1′8′, G1′-11′ and G1′-16′ are significantly more potentthan the shorter G1′-6′ and G1′-7′ (FIG. 17). This observation fits verywell with the crystal structure of PapD complexed with G1′-19′ whichshows that the C-terminal 8 residues in G1′-19′ are hydrogen bonded toPapD. The shorter peptides G1′-6′ and G1′-7′ are unable to fulfil thishydrogen bonding pattern and are therefore less active inhibitorypeptides (FIG. 18). The replacement series reveals that residues 4′, 5′and 6′ in G1′-8′ form important contacts with PapD, since theirreplacement results in less active inhibitory peptides (FIG. 18). Thedeletion series again indicated an important role for residues 4′, 5′and 6′ in G1′-8′ for the complex formation with PapD (FIG. 19). However,the results obtained with the deletion series did not support the“zippering” hypothesis according to which the members of the deletionseries would show an increasing inhibitory potency as the deletion ismoved form the C-terminus towards the N-terminus of G1′-81.

As appears from these preliminary results, the results obtained in theassay exhibit large deviations from the mean in each experiment. Asdiscussed herein, one reason for this might be the slow kinetics ofbinding between PapD and correctly folded pilus subunit proteins andanalogues of such correctly folded subunit proteins. It is thereforecontemplated to modify the assay by introducing denaturing influencespowerful enough to at least partially unfold the pilus subunit(analogues). It is expected that this will reduce the deviations in theassay results. Another reason for the large deviations might be bindingof the peptides to BSA used in the ELISA. Therefore, the replacement ofBSA with other macromolecules will be investigated.

In case the “zipper mechanism” for peptide binding to PapD can beconfirmed, peptide chaperone binding can be optimized using a limitedsynthetic peptide library in which Pro-1′, Phe-2′, Leu-4′, Met-6′ andMet-8′ of the PapG 8-mer are replaced by the hydrophobic amino acidsVal, Leu, Ile, Met, Phe, Trp, Tyr and His. Non-natural amino acids suchas D-amino acids and N-methylated amino acids, as well as amino acidscontaining aliphatic and various aromatic side chains, will also beincorporated in the chemical library. Optimal amino acid combinationsfor these five positions will be used in the synthesis of the chaperoneinhibitors described below in this Example. If the “zipper mechanism” isnot confirmed, the outlined approaches will-be applied to residues shownto be important for binding to PapD.

Chaperone inhibitors that form covalent bonds to the chaperone afterdocking into its active site will be developed. The crystal structure ofthe PapD-peptide complex shows that the C-terminal carboxyl group of thepeptide is hydrogen bonded to Lys-112 and Arg-8 in the chaperone andthat the side chain of Val-5′ in the peptide is close to the side chainamino group of another Lys in PapD. Introduction of reactive groups suchas alkyl halides, aldehydes, acid halides and active esters in thesepositions of the optimized 8-mer peptide will lead to the formation ofcovalent bonds to the lysines in PapD, and the peptide derivatives thusconstitute high affinity inhibitors. The inhibitors are based on nonameror shorter peptides in which residue 5 (from the C-terminus) and/or theC-terminal carboxyl group have been modified as indicated:

Replacements for residue 5 from the C-terminus:

Replacements for the C-terminal COOH-group

—COX, X as above

—CH₂Y, Y as above

Such interactions between aldehydes and lysine side chains have givenpotent drug candidates for sickle-cell anaemia.

In the PapD-peptide crystal, the peptide forms an extension of a β-sheetin the chaperone. Restrictions that give the peptide a β-sheet likeconformation will therefore result in a favourable change in the entropyof binding. Conformationally restricted peptides constituting miniaturecyclic β-sheets or peptides having the side chains of amino acidsseparated by one amino acid covalently linked to each other will beprepared based on the optimized 8-mer peptide. Peptides with covalentlylinked side chains will have the three consecutive amino acids replacedby the fragments shown below:

AA=any natural or nonnatural amino acid

R_(a),R_(b)=all possible combinations of H, Me, Et, C₃H₇, C₄H₉, C₆H₅

AA=any natural or nonnatural ammo acid

R_(a),R_(b)=all possible combinations of H, Me, Et, C₃H₇, C₄H₉, C₆H₅

Interesting inhibitors will be co-crystallized with chaperones, and thecomplexes will also be investigated by NMR spectroscopy.

Peptides and peptide mimetics used as drugs can be rapidly metabolizedby proteolytic enzymes that cleave the peptide bonds. Chymotrypsinselectively cleaves the peptide bond on the carboxyl side of amino acidswith aromatic and large hydrophobic side chains. In the PapG 8-mer, theMet-8′-Thr-7′, Met-6′-Val-5′ and Phe-2′-Pro-1′ amide bonds are thereforeespecially sensitive to proteolysis and should be replaced bymetabolically stable peptide isosters. Examples of such peptide isostersare-given below:

Replacements for the

fragment:

Example 8

The Role of Periplasmic Chaperones in the Infectivity of Bacteria WhichAdhere By the Means of Pili

The specific role of chaperone-assisted pilus assembly in virulence canbe determined by comparing the adherence and pathogenicity of piliatedwild type and isogenic mutants that are non-piliated due tosite-directed mutations in the active site of the chaperone. Mutationsin residues such as Arg-8 and Lys-112 that constitute the subunitbinding site of PapD can be recombined into the bacterial chromosome ofthe clinical isolate strain DS17 using the same method that has beensuccessful to introduce point mutations into the papG gene in thechromosome of the same strain. E. coli strain DS17 spread epidemicallywithin a neonatal ward, causing several cases of pyelonephritis. Inaddition, the strain has been found to cause acute kidney infections ina cynomolgus monkey pyelonephritis model. DS17 contains one pap genecluster with a papG gene expressing a typical papG adhesin. It alsoexpresses typical type 1 pili containing the mannose-binding adhesin,FimH. Type 1 pili have been suggested to be important virulencedeterminants in cystitis. Therefore, site-directed mutants in thesubunit binding site of the FimC chaperone should also be generated totest this concept.

It is contemplated that a compound that binds the chaperone active sitewould block pilus assembly and thus prevent attachment. If this conceptis valid, then well-defined chaperone mutants such as in Arg-8 andLys-112 should abolish receptor binding, too. The receptor bindingactivity of various chaperone mutants should be measured in the receptorbinding ELISA assays described in examples 2 and 6. To measure type 1pilus-mediated binding, mannose will be linked to the wells of themicrotiter plates. Adherence of various FimC and PapD chaperone mutantsto the immobilized receptors will be quantitated using antibodies to E.coli DS17 in an ELISA experiment. Furthermore, the ability of smallPapD-binding peptides to block pilus assembly in DS17 and, thus, toabolish receptor binding will be tested. Strain DS17 will be grown inthe presence of short PapD-binding or non-binding peptides and thentested for their ability to bind receptor in the ELISA experiment. Theseexperiments will make important strides at validation the concept thatanti-chaperone inhibitors would prevent bacterial attachment in vitro.

Then, the role of chaperone-assembled adhesins in causing diseases willbe established The causality between E coli expressing P pili andpyelonephritis has until recently been based solely on epidemiologicaldata. It has been shown that the P piliated strain DS17 causespyelonephritis to occur in the normal urinary tract of cynomolgusmonkeys. A mutation in the papG gene of strain DS17 was generated byallelic replacement to create the derivative strain DS17-8 to test therequirement of the PapG adhesin in causing the disease. DS17-8 containsone base pair deletion after codon 37 in papG resulting in theexpression of P-pili lacking the PapG tip adhesin. This mutant wasunable to bind the globoside receptor in vitro and was unable to bind tokidney tissue from humans or cynomolgus monkeys in our in situattachment model. The virulence between DS17 and its papG mutant DS17-8in the cynomolgus monkey was compared. To study the role of the PapGadhesin in pyelonephritis, five monkeys were infected with E. colistrain DS17 whereas six monkeys received mutant strain DS17-8 via acytoscopically inserted ureteral catheter, and it was shown that therewas a significant difference between the two groups. The monkeysreceiving wild type strain DS17 has a mean bacteruria of 21 dayscompared to 6.8 days for those monkeys receiving the mutant strainDS17-8. Renal clearance was also significantly lower for the wild typestrain and renal function was significantly reduced in the monkeysreceiving the wild type strain but not in those monkeys receiving theisogenic papG mutant strain. Pathologic evaluation of the infectedkidney confirmed the functional studies showing significantly lessinflammation and pathologic changes in the mutant group as compared tothose receiving the wild type.

Therefore, it is concluded that the Galα1-4Gal binding PapG adhesin atthe tip of P pili is required for pyelonephritis to occur. in the normalurinary tract of primates. Up to now, no such direct demonstration forthe role of a pilus associated adhesin in a specific bacterial infectionhas been made.

Interestingly, in the experiments described above, no evidence was foundthat the PapG adhesin was required for colonization of the lower urinarytract or for the development of acute cystitis. Both strains colonizedthe vagina, persisted in the intestine and caused bladder infection.Thus, although PapG was a critical virulence determinant in causingpyelonephritis, it did not appear to be crucial in cystitis. However,this did not exclude the possibility that other components of the Ppilus or that another type of pilus, such as type 1, was required forcystitis.

These hypotheses will be investigated by testing the virulence ofisogenic DS17 mutant strains containing site directed mutations in Arg-8of PapD or FimC. The mutants will presumably be defective in theirability to assemble P and type 1 pili. If the papD mutant isnon-virulent in the monkey cystitis model, it may indicate that somecomponent of the P-pilus other than PapG is essential for E. coli togenerate bladder infection. For example, the major component of the tipfibrillum, PapE, has been shown to bind fibronectin and fibronectincould be an important factor in cystitis. Similarly, it the fimC mutantis non-virulent, it would confirm the role of the mannose-binding type 1pili in causing cystitis. The ability of these point mutations toabolish the virulence of DS17 in causing either pyelonephritis orcystitis would validate the therapeutic potentials for a chaperoneinhibitor.

Example 9

Identification of the Motif of Binding Between PapD and K1′-19′WT

In order to confirm the generality of the binding mode for C-terminalpeptides of pilin subunits to PapD as observed in the PapD-G1′-19′WTcrystal structure (see Example 1) a second PapD-peptide complex wasinvestigated by X-ray crystallography. Since tight binding had beenobserved for the peptide derived from the wild-type C-terminal 19amino-acids of PapK (K1′-19′WT, SEQ ID NO: 18, and numbered here fromthe C-terminal Arg-1′ to the N-terminal Lys-19′) it was decided to usethis as the peptide for the second PapD complex.

Obtaining Material; Protein & Peptide

PapD was prepared as previously described (Holmgren et al., 1988) andobtained from Dr. Scott Hultgren, Dept of Molecular Microbiology,Washington University School of Medicine, St. Louis USA. The peptideK1′-19′WT was prepared by Fmoc solid phase synthesis, purified byreversed phase HPLC and obtained from Dr. Jan Kihlberg, Dept. ofChemistry, University of Lund, Lund, Sweden.

Crystallisation of PapD-peptide Complex

After a number of different experimental conditions had been exploredaround those previously used to obtain PapD-G1′-19′WT crystals the bestcrystals of the PapD-K1′-19′WT complex were grown by vapour diffusionagainst 20% PEG8000, 0.1 M MES pH 6.5. The crystallisation dropcontained equal volumes of reservoir and protein solution. The proteinsolution (15 mg/ml) contained a 1:1 molar ratio of PapD to peptide in 20mM MES pH 6.5 with 1.0% β-octyl glucoside (β-OG).

These crystals were mounted inside sealed quartz-glass capillary tubesand initially characterised by examining them on a X-ray precessioncamera. From standard analysis of such images it was determined that theabove-mentioned crystals have a orthorhombic space group, C2221 (thusdiffering from the PapD-G1′-19′WT crystals which were C2), with celldimensions a=57.1 Å, b=153.2 Å, c=135.4 Å and α=β=γ=90°, 2 molecules inthe asymmetric unit and diffract to 2.7 Å resolution on a lab X-raysource with rotating anode and Cu K_(α) target.

Collection & Processing of Experimental Data

The intensity data for the PapD-K-peptide crystals were collected on aR-AXIS II area-detector system (from R-AXIS) at Symbicom AB., Uppsala.All data were obtained from a single crystal and processed initiallywith the DENZO software ware package (Otwinsky, 1993). Merging andscaling of the data, however, was carried out using ROTAVATA andAGROVATA from the CCP4 package (CCP4, 1979). The final data setcontained 15,989 independent reflections with an Rsym of 8.7% for databetween 20.0 and 2.7 Å resolution.

Solution of Three Dimensional Structure

The structure of the complex was solved by the standard method ofmolecular replacement using the program XPLOR (Brunger, 1992). Thesearch model used was the refined 2.0 Å resolution structure of PapD(Holmgren and Bränden, 1989. Using 8.0 to 4.0 Å resolution data theself-rotation function again gave a clear non-crystallographic two-foldaxis. The top peaks in the translation functions also gave the correctsolutions. After the translation functions the R-factor was 36.7% for8.0 to 4.0 Å resolution data. Subsequent rigid body refinement in whichall 4 domains of the 2 PapD molecules in the asymmetric unit wereallowed to refine independently resulted in an R-factor of 33.6% for thesame data.

Examination of an |Fo|−|Fc| electron density map at this stage using thegraphics program O (Jones and Kjeldgaard, 1994) showed clear densitycorresponding to the K-peptide in the PapD cleft and running along thesurface of the protein in an analogous fashion to that found for theG-peptide. The orientation of the peptide was easily determined from theelectron density, but initially only the final 12 C-terminal amino-acidsof the peptide could be modelled into density.

Refinement and Analysis of Structure

Simulated annealing refinement with XPLOR (Brunger, 1992) was initiatedat this stage. Several additional cycles of model building andrefinement were carried out with a further 2 peptide amino-acids beingadded to the N terminal end of the peptide to yield an R-factor for thecurrent model of 19.2% for 8.0 to 2.7 Å resolution data. The model atthe present stage of refinement (which contains no water molecules anddoes not include the first 5 N-terminal amino-acids of the peptide) hasroot-mean square (rms) deviations from ideal geometry of 0.019 for bondslengths and 3.8° for bond angles.

Despite the two PapD-peptide complexes being solved in different spacegroups, their overall structures are found to be highly similar with thepeptide interacting with PapD in a manner essentially identical to thatpreviously seen for the PapD-G1′-19′WT structure. Thus the K1′-19′WTpeptide is again seen to bind in an extended conformation with theC-terminal Arg-1′ anchored within the inter-domain cleft and subunitbinding site. Hydrogen bonds are also formed between the peptide carboxyterminus and two invariant positively charged residues of PapD, Arg-8and Lys-112. The K1′-19′WT peptide then run along the surface of theN-terminal domain, forming a parallel β-strand interaction with strandG1. In this way between 9 mainchain hydrogen bonds are formed betweenresidues 10′ to 2′ of the peptide and Val-102 to Lys-110 of PapD andthus extending the β-sheet of PapD out into the peptide.

In addition, a dimer association is observed between the twoPapD-peptide complexes within the unit cell similar to that seen in thePapD-G1′-19′WT crystal structure. Again the β-sheet is extended as aresult of a non-crystallographic 2-fold symmetry which places a secondPapD-peptide complex adjacent to the first such that the two boundpeptide chains interact as anti-parallel β-strands with a mixed β-sheetagain being created between the two complexes involving a total of 10β-strands. The only major difference between the two PapD-peptidecomplexes is the fact that in the PapD-K1′-19′WT structure the peptidesof the non-crystallographically related complexes are positioned tworesidues closer to the COOH-terminus of its partner. Thus while eighthydrogen bonds are formed between the peptides in the PapD-G1′-19′WTcomplex a total of 10 are observed in the PapD-K1′-19′WT

Apart from the C-terminal residues Tyr-2′ and Arg-1′ there are againrelatively few contacts between the sidechains of the peptide and PapD.The major interactions are provided by the mainchain hydrogen bonds tothe G1 strand. There are, however, a number of hydrophobic interactionswithin the β-sheet, in particular between the peptide's Tyr-6′ withIle-105 and Leu-107 of strand G1.

Example 10

Identification of a Second Binding Site in PapD and Development of NewAssays

Introduction

A chaperone binding assay was developed to delineate PapD-PapGinteractions using MBP/G fusions, containing increasing lengths of theCOOH-terminus of PapG. The ability of PapD to bind to PapG truncateproteins missing increasing lengths of the COOH terminus of PapG wasalso examined.

Experimental Procedures

Bacterial Strains

The E. coli strain HB101 (Maniatis et al., 1982) was used as a hoststrain in the studies involving the MBP/G fusion proteins. Strain DH5αwas used as the host to construct MBP/G fusion proteins (Hanahan, 1983)and PapG truncates. KS474 (degP::kan) (Strauch et al., 1989) was kindlyprovided by J. Beckwith and used for expression of the PapG truncateproteins.

Plasmid Construction

The E. coli expression plasmid pMAL-p2 (new England Biolabs, Beverly,Mass., USA) was used for the construction of pMAL-p4, pMAL/G1′-19′,pMAL/G1′-81′ and pMAL/G1′-140′ using procedures essentially fromManiatis (Maniatis et al. 1982). The malE gene encoded by pMAL-p2 has alacZα sequence fused to the 3′ end. Plasmid pMAL-p2 was digested withSalI and filled in with Klenow (Maniatis et al., 1982). The fragment wasreligated and the resulting plasmid called pMAL-p4 had a stop codonbetween the malE and lacZα sequences. Plasmid pMAL-p4 was digested withHindIII and filled in with Klenow and the resulting fragment was ligatedwith a ClaI linker, 5′CCATCGATGG3′ (New England Biolabs, Beverly, Mass.,USA) to produce plasmid pMAL-p5. Primer 23969(5′CCCCCCTGCAGATCAGATTAAGCAGCTACCTGC3′, SEQ ID NO: 23) and primer 23557(5′CCCCTGCAGTAAAAATATCTCTGCTCAGAAATAC3′, SEQ ID NO: 24) and primer 23559(5′CCATCGATGAACAGCCAGTCAGATAATC3′, SEQ ID NO: 25) were used forPolymerase Chain Reactions (PCR) utilizing pPAP5 (Hull et al., 1981;Lindberg et al., 1984) as the template. The DNA fragments encoding theregion of amino acid residues 1′-81′ and 1′-140′0 of the PapG wereobtained from the PCR reactions using primers 23557 and 23559; andprimers 23969 and 23559, respectively. The amplified DNA fragments werepurified and restricted with PstI and ClaI and ligated into PstI andClaI restricted pMAL-p5 fragment to construct pMAL/G1′-81′ andpMAL/G1′-140′, respectively. To create pMAL/G1′-19′, two nucleotideoligomers (5′GGAAAGAGAAAACCCGGGGAGCTATCTGGTTCTATGACTATGGTTCTGAGTTTCCCCTGAT3′, SEQ ID NO: 26 and5′CGATCAGGGGAAACTCAGAACCATAGTCATAGAACCAGATAGCCCCGGGTTTTCTCTTT CCTGCA3′,SEQ ID NO: 27) were chemically synthesized and annealed. The annealedDNA fragment containing the sequence of the carboxyl terminal 1′-19′amino acid residues of PapG was ligated to PstI-ClaI pMAL-p5 fragment toproduce pMAL/G1′-19′. All the constructs used in this study wereconfirmed by DNA sequencing analysis using Sequenase Version 2.0according to the manufacturer's directions (USB, Cleveland, Ohio, USA).pHJ14 (PapG3) was created by digestion of pHJ8 (wt PapG in P_(tac)promoter plasmid, pMM66) with BamHI and BglII, removing approximately500 nucleotides, purification of the large fragment and religation.pHJ23 (PapG 2) was created by first cloning papG into pUC19 at EcoRI andBamHI, digesting with HincII to remove the last approximately 300nucleotides and religating. The EcoRI-HincII fragment was then clonedinto pMMB66 to create pHJ23.

Induction and Partial Purification of MBP/G Fusions

Strains carrying MBP or MBP/G fusion gene plasmids were induced in LBbroth culture with 1 mM isopropylthiogalactoside (IPTG). Periplasmicextracts were prepared as described (Slonim et al., 1992). One tenthvolume of 10× phosphate-buffered saline (PBS, 120 mM NaCl, 2.7 mM KCl,10 mM Na₂HPO₄ and 2 mM KH₂PO4, pH 7.4) was added to the periplasmicextract. Amylose resin was then added to the periplasmic extracts at a1:5 ratio. The mixture was rocked at 4° C. overnight and the beads weresubsequently washed 5 times with PBS. MBP or MBP/G fusion proteins wereeluted with 20 mM maltose in PBS by rocking at 4° C. for an hour.Protein concentrations were determined using the Bio-Rad DC proteinassay kit (Bio-Rad Laboratories, Hercules, Calif., USA). The full lengthfusion protein concentration was further quantitated by the Coomassiestained SDS-PAGE gel with known concentrations of bovine albumin (BSA)as the standards followed by densitometry scanning.

Characterization of Interactions Between PapD and MBP/G Fusion Proteins

The MBP or MBP/G fusion preparations from HB101/pLS101/pMAL-p4,HB101/pLS101/pMAL/G1′-19′, HB101/pLS101/pMAL/G1′-81′ andBH101/pLS101/pMAL/G1′-140′ were applied to isoelectric focusing (IEF) pI3-9 gels (Pharmacia Phast System, Pharmacia, Sweden) or 12.5% SDS-PAGEfollowed by silver staining, Coomassie blue staining or immunoblottingwith anti-PapD antiserum as described (Kuehn, et al., 1991).

The stability of the PapD-MBP/G1′-140′ complex in urea was determined byincubating 1 μg partially purified MBP/G1′-140′ preparation fromHB101/pLS101/pMAL-G140 in 0-5 M urea for 5 minutes at 25° C. (Kuehn etal., 1991). The proteins were analyzed by IEF (pI 3-9) with silverstaining. The PapD-MBP/G1′-140′ complex on the IEF gel was quantified bydensitometry (Digital Imaging System, Is-1000).

The interaction between PapD and MBP/G fusion in vitro was assayed byincubating purified PapD (1 μg) and amylose bead affinity purified MBP/Gfusion protein (1 μg) in 4 μl PBS for 30 minutes and then applied to IEF(pI 3-9), followed by silver staining. Another in vitro assay that ismore quantitative is the ELISA described below.

Characterization of the Interaction Between PapD and PapG Truncates

PapD and the PapG-truncates were co-expressed in KS474, the truncateswere induced at OD₆₀₀ 0.6 with 0.5 mM IPTG and PapD under the control ofthe arabinose promoter was induced with 0.2% arabinose. After one hourinduction, periplasmic extracts were prepared and rocked with Gal α(1-4)Gal beads overnight at 40° C. and eluted with 15 μg/ml Gal α(1-4)Gal-TMSET solution as described previously (Hultgren et al., 1989; Kuehnet al., 1991). The eluates were analyzed by acidic native geleletrophoresis (Striker et al., 1994) followed by western blotting usinganti-PapD and anti-PapG antiserum.

Hemagglutination Titers

HB101/pFJ3 harbouring various MBP/G fusion plasmids was passaged onTrypticase Soy Agar (TSA, Becton Dickinson, Cockeysville, Md., USA)three times to induce P-pili expression. At the last passage, theexpression of MBP/G fusions was induced on TSA containing 100 μM IPTG.The cells from different strains were then collected and the HA titerswere determined as described (Jacob-Dubuisson et al., 1993b).

Pili Preparation and Quantitation

The cells obtained from TSA plates were collected. Pili were preparedfrom the same amounts of cells using the procedure described previously(Jacob-Dubuisson, et al., 1993b). These pili preparations were boiled inLammeli sample buffer with 4 M urea, then analyzed on Coomassie bluestained SDS polyacrylamide gels. The relative amount of piliation wasquantified by densitometric scanning of the PapA bands.

ELISA Assay

Stock solutions of MBP/G fusion proteins in PBS were diluted to 40pmol/50 μl with PBS. The solutions of the 40 pmol/50 μl MBP/G fusionProteins were serially diluted in PBS and 50 μl of each solution wereused to coat the wells of a 96-well Nunc Immunoplate (Inter Med, DK-4000Roskilde, Denmark) overnight at 4° C. The wells were then washed withPBS and blocked with 200 μl of 3% BSA in PBS for 2 hours at 25° C. Theplates were washed vigorously three times with PBS and incubated with 50μl of PapD diluted at 50 pmol PapD in 50 μl 3% BSA-PBS for 45 minutes at25° C. After three washings with PBS, the wells were incubated with a1:500 dilution of rabbit anti-PapD antiserum in 3% BSA-PBS for 45minutes at 25° C. After three washings with PBS, the wells wereincubated with a 1:1000 dilution of goat antiserum to rabbit IgG coupledto alkaline phosphatase in 3% BSA-PBS for 45 minutes at 25° C. Afterthree washings with PBS and three washings with developing buffer (10 mMdiethanolamine, 0.5 mM MgCl₂), 50 μl of filtered 1 mg/ml p-nitrophenylphosphate (Sigma, St. Louis, Mo., USA) in developing buffer was added.The reaction was incubated for 1 hour in the dark at 25° C. and theabsorbance at 405 nm was read.

For assaying the second site peptides, all the peptides were dissolvedin dimethyl sulfoxide (DMSO, Sigma, St. Louis, Mo., USA) to a finalconcentration 2 mM. The stock solutions were diluted to a properconcentration in PBS and coated overnight on microtiter wells at 4° C.All the peptide solutions were adjusted to the same DMSO concentration.The subsequent steps followed the procedure described above.

Results

MBP/G Fusions

To identify determinants on PapG essential for PapD interaction weconstructed three in frame fusions between the MalE gene, encodingmaltose binding protein, and sequences encoding the COOH-terminalresidues 1′-19′, 1′-81′ and 1′-140′ of PapG (FIG. 20). The resultingchimeric proteins were called MBP/G1′-19′, MBP/G1′-81′ and MBP/G1′-140′,respectively, to indicate the regions of PapG present in the MBP/Gfusions. MBP/G1′-19′ was created since PapD was recently shown to bind apeptide consisting of the C-terminal 19 amino acids of PapD in vitro(Kuehn et al., 1993). MBP/G1′-81′ and MBP/G1′-140′ were created to testthe requirement of the disulfide bridge (C196-C228 in PapG) forrecognition by PapD. A similarly located disulfide bond is found invirtually all pilus subunits (Simons et al., 1990). MBP/G1′-81′ lacksthe two cysteine residues while MBP/G1′-140′ contains the disulfide bond(FIG. 20).

PapD-MBP/G Interactions In vivo

The ability of PapD to bind to the MBP/G fusions was investigated usingamylose affinity chromatography (Kellermann and Ferenci, 1982). PapD wasco-expressed from plasmid pLS101 (Slonim et al., 1992) with each of theMBP/G fusions. Periplasmic extracts from each strain containing theMBP/G proteins and PapD were subjected to amylose affinitychromatography and the eluates analyzed by SDS-PAGE (FIG. 21A) and bywestern blotting with anti-PapD antisera (FIG. 21B). In this assay,co-elution of PapD signified the ability of PapD to interact with theMBP/G protein. The western blot revealed that PapD co-eluted with allthree fusion proteins (FIG. 21B, lane 2, 3 and 4) but not with the MBPcontrol (FIG. 21A and 21B, lane 1). However, PapD interacted muchstronger with the MBP/G1′-140′ fusion as can be seen in the western blot(FIG. 21B, lane 4) as well as in the coomassie blue stained gel of theeluates (FIG. 21A, lane 4). Thus, PapD interacted strongly with theMBP/G1′-140′ protein and only weakly with the MBP/G1′-19′ andMBP/G1′-81′ proteins.

Analysis of the eluates on silver stained isoelectric focusing (IEF)gels revealed that the PapD-MBP/G1′-140′ complex migrated at anisoelectric point (pI) of 5.2 (FIG. 21C, lane 4) which was intermediatebetween the pIs of MBP/G1′-140′ (˜4.4) and PapD (˜9.1). This band wasconfirmed to contain both PapD and MBP/G1′-140′ by excising theunstained band, applying the material to SDS-PAGE and analyzing it bywestern-blotting using anti-PapD and anti-MBP antisera (FIG. 21D). Itwas not possible to detect stable complexes in the eluates of MBP,MBP/G1′-19′ or MBP/G1′-81′ when co-expressed with PapD (FIG. 21C, lane1, 2 and 3).

The stability of the PapD-MBP/G1′-140′ complex was measured as afunction of urea concentration in the presence of 15 MM DTT. Both thePapD-PapG and PapD-MBP/G1′-140′ complexes behaved similarly under theseconditions and were dissociated after incubation in 2M urea (data notshown). By these criteria, the PapD-MBP/G1′-140′ complex was as stableas the PapD-PapG complex.

Expression of MBP/G Proteins Inhibit Pilus Formation

PapD is essential for P-pilus assembly (Hultgren et al., 1991). Adecrease in the concentration of PapD in the periplasm has been shown tocause a concomitant decrease in piliation (Slonim et al., 1992). Theability of the MBP/G fusions to block pilus formation by inhibitingchaperone-subunit complex formation when co-expressed in trans with thepap operon was tested. Plasmids pMAL-p4, pMAL/G1′-19′, pMAL/G1′-81andpMAL/G1′-140′ were transformed into the strain HB110/pFJ3 which containsthe pap operon under the control of its own promoter in the vectorpACYC184 (Jacob-Dubuisson et al. 1993b). Each MBP/G fusion protein waslocalized in the periplasm as determined by SDS-PAGE and westernblotting with anti-MBP antisera (data not shown). Hemagglutination (HA)assays were performed on the cells co-expressing the pap operon witheither MBP (pMAL-p4) or with each of the MBP/G fusions. Co-expression ofthe MBP/G1′-140′ fusions with the pap operon decreased the HA titer 30fold compared to the MBP control (see table 3).

TABLE 3 MBP/G fusions inhibit P pilus formation NBP/proteins^(a) MBPMBP/G1′-19′ MBP/G1′-81′ MBP/G1′-140′ HA titer^(b) 128  64  82  4 Piliation percentage^(c) 100% 50% 40% 10% ^(a)Plasmids pMAL-p4,pMAL/G1′-19′, pMAL/G1′-81′ and pMAL/G1′-140′ encoding MBP, MBP-G1′-19′,MBP-G1′-81′ and MBP-G1′-140′, respectively, were transformed into thestrain HB1010/pFJ3 and the expression of pap operon and each of theMBP/G fusions was induced in each strain. ^(b)Highest dilution of cellsuspension yielding detectable HA ^(c)The pili preparation were analyzedon Coomassie blue stained gels and the PapA bands were scanned by adensitometer. The relative piliation was determined by equating thedensitometric value of PapA to 100% of the control.(HB101/pFJ3/pMAL-p4).

Co-expression of the MBP/G1′-81′ and MBP/G1′-19′ fusions with the papoperon had only weak effects on the HA titer (Table 3). Co-expression ofMBP/G1′-140′ with the pap operon reduced the amount of pili that couldbe purified from the cells by 90% whereas MBP/G1′-19′ and MBP/G1′-81′reduced the amount of pili formed by about 50% compared to MBP alone(Table 3). Electron microscopy confirmed that cells expressing theMBP/G1′-140′ fusion had little or no pili compared to the fully piliatedcells co-expressing MBP (data not shown).

We hypothesized that the co-expression of the MBP/G1′-140′ fusion withthe pap operon inhibited pilus formation by titrating PapD away from thesubunits thus driving subunits down dead end pathways of aggregation andproteolytic degradation (Hultgren et al., 1989; Holmgren et al., 1992).This hypothesis was tested as follows. pMAL-p4, pMAL/G1′-19′,pMAL/G1′-81′ or pMAL/G1′-140′ were transformed into BH101 carrying pFJ22(Jacob-Dubuisson et al., 1994). Plasmid pFJ22 encodes papDJKEFGA underthe control of the P_(tac) promoter. In HB101/pFJ22, pilus subunitsaccumulate in the periplasmic space due to the absence of the usher,PapC. The effect of expressing each fusion on the fate of each pilussubunit type was determined by western blotting (FIG. 22). A significantdecrease in the amount of PapG, PapA and PapF in cells co-expressing theMBP/G1′-140′ fusion was detected arguing that the MBP/G1′-140′ fusionblocked chaperone-subunit complex formation by interacting with PapD andtrapping it away from pilus subunits. There was no effect on PapK andPapE stability for reasons that are not understood. Nevertheless, thefusion was capable of blocking pilus formation by interfering with theformation of critical chaperone-subunit complexes.

In vitro Chaperone Binding Assays

PapD binding to the MBP/G fusion proteins was further investigated usingtwo different in vitro assays. In the first assay, purified MBP/G fusionproteins or MBP, were incubated with PapD and complex formation wasanalyzed on silver stained IEF gels. PapD bound to the MBP/G1′-140′fusion protein and formed a stable complex that migrated to an identicalpI (5.2) as that seen for the complex formed in vivo (FIG. 23A, lane 4).In contrast, no complexes between PapD and MBP/G1′-81′, MBP/G1′-19′ orthe MBP control were detected (FIG. 23A, lane 1, 2 and 3). In the secondassay, we investigated the ability of PapD to bind to the threedifferent MBP/G fusion proteins immobilized on microtiter wells andquantitated the interactions in an enzyme-linked immunosorbent assay(ELISA) using anti-PapD antiserum (FIG. 23B). PapD bound weakly toMBP/G1′-19′, slightly better to MBP/G1′-81′ but very strongly to theMBP/G1′-140′. PapD did not bind the MBP control. The dramatic increasein the affinity of PapD for the MBP/G1′-140′ fusion as compared to theMBP/G1′-81′ and MBP/G1′-19′ fusions, suggested that residues 81′ to 140′in PapG were critical for strong PapD binding.

The invariant cleft residues Arg-8 and Lys-112 in PapD have been shownto form a molecular anchor in the chaperone cleft necessary for bindingpilus subunits (Slonim et al., 1992; Kuehn et al., 1993). Mutations inthese residues abolished or greatly reduced the ability of PapD to bindsubunits and mediate pilus assembly (Kuehn et al., 1993). Arg-8A andLys-112A mutant PapD proteins demonstrated a greatly reduced ability tobind to the MBP/G1′-140′ fusion protein both in vivo and in vitro (datanot shown). These results argue that the interactions between PapD andthe MBP/G1′-140′ fusion are biologically relevant.

The Upstream Site is an Independent Binding Determinant

We tested the ability of PapD to bind three PapG COOH-terminal truncateproteins (shown in FIG. 20) in order to delineate the limits of theupstream PapD binding site and to test whether it was capable offunctioning as an independent site. The truncate proteins wereco-expressed with PapD in strain KS474 (Strauch et al., 1989) whichcarries a kanamycin cassette in the degP locus (degP41 mutant). Both thePapG2 and PapG3 truncates were expressed and stable in KS474, howeverthe PapG1 truncate (removing the last 14 residues) underwent limiteddegradation (data not shown). Complex formation was assayed by analyzingperiplasmic extracts on acid native polyacrylamide gels (Striker et al.,1994). Anti-PapD and anti-PapG antisera was used to detect the PapD-PapGcomplexes after western blotting (FIG. 24). PapD-PapG complexes areresolved from PapD alone on these gels due to their differences in size,shape and charge (Striker et al., 1994) PapD formed a complex withfull-length PapG as well as with the PapG2 truncate (FIG. 24A, lanes 2and 3). The complex band was also recognized by anti PapG antiserum(FIG. 24B, lanes 2 and 3). PapD did not form a complex with the PapG3truncate (FIG. 24A & B, lane 4) which terminates at amino acid 145. Thisinformation delineates an endpoint for the second PapD binding site (asindicated in FIG. 20).

Combining the PapG truncate and the MBP/G fusion data (shown in FIG. 20)suggested that the second site on PapG recognized by PapD resided in aregion between residues 117′ to 141′ of PapG. Four overlapping peptideswere synthesized corresponding to the region between residues 120′ to156′ and tested for their ability to bind PapD in an ELISA assay. Such astrategy proved successful in studying the COOH-terminal PapD bindingsite (Kuehn et al., 1993). The peptide corresponding to residues 125′ to140′, but not the other three peptides, bound to PapD in the ELISA (FIG.25). These data argue that PapD recognizes two surfaces on PapG. PapDforms a beta strand zippering interaction with the COOH-terminus butalso recognizes a region containing residues 125′-140′.

Discussion and Conclusions

In conclusion, a chaperone binding assay was developed using fusions ofthe carboxyl terminus of PapG to Maltose Binding Protein (MBP/G fusions)to investigate whether chaperone-subunit complex formation requiresadditional interactions. PapD bound strongly to an MBP/G fusioncontaining the C-terminal 140 amino acids of PapG (MBP-G1′-140′) butonly weakly to the MBP-G1′-81′, arguing that the region between theC-terminal residues 81′ and 140′ contains additional information that isrequired for strong PapD-PapG interactions. PapD was further shown tointeract with a PapG C-terminal truncate containing residues 117′-314′(corresponding to a truncate consisting of the first 198 N-terminalamino acid residues of PapG), but not with a truncate containingresidues 170′-314 (corresponding to a truncate consisting of the first145 N-terminal amino acid residues of PapG).

These results together suggest that a second, independent PapDinteractive site exists which does not interact with the C-terminus ofthe pilus subunit. This assumption is further confirmed by the fact thatone of four peptides overlapping the second site region of PapG wasrecognized by PapD. The last result also rules out that the function ofthe upstream site was to exert secondary effects on the COOH terminalchaperone binding site since this region was independently capable offacilitating PapD binding.

Unlike what is true for the native subunits it was demonstrated thatMBP-G1′-140′ is capable of folding even in the absence of PapD andremain soluble in the periplasmic space; this folding involves theformation of an intramolecular disulphide bond. This is consistent withearlier data showing that the subunits themselves are highly folded whenbound to PapD (Kuehn et al., 1991; Striker et al., 1994). However, it isnot yet known whether PapD binds to subunits in vivo after they fold orwhether folding occurs in the context of an. interaction with PapD.

Finally, the expression of the MBP/G fusions in cells producing P piliinhibited pilus assembly to varying degrees by binding to PapD andpreventing chaperone-subunit complex formation. The ability of thefusions to block pilus assembly increased as the length of the fusionprotein increased. MBP-G1′-140′ was a strong inhibitor of pilus assemblywhile MBP-G1′-81′ and MBP-G1′-19′ were weak inhibitors. This isconsistent with the finding that residues 140′-81′ contain a regionnecessary for strong interactions between PapD and PapG.

Recent research by the inventors has further revealed that thesimultaneous expression in vivo of PapG and a modified PapD polypeptidefree of domain 1 in a degP41 strain (KS474) expressing this truncatedPapD results in 1) suppression of PapG toxicity in this cell line and 2)efficient partitioning into the periplasmic space of PapG. However, thepilus subunit is not correctly folded after release from PapD.

It is thus suggested by the inventors, that the binding between pilussubunits and PapD takes place by the pilus subunit binding to domain 1and 2 of PapD. The binding between the subunit and domain 1 is importantfor the correct folding of the subunit, and the binding between thesubunit and domain 2 is important for the transport of the subunit outinto the periplasm. Whether domain 2 of PapD also participates in thefolding of subunits is not known.

Since the above results are strong indicators of the existence of atleast one binding site apart from the one involving Arg-8 and Lys-112 ofPapD, and since it seems that this novel binding site is also veryimportant in the net interaction between PapD and the pilus subunits, itis contemplated that also effecting this binding site will have theantibacterial effects described herein.

It is thus the plan to elucidate the motif of binding between thissecond binding site of PapD in manners similar to those describedherein, and it is further the plan to design/identify compounds capableof interacting with this second binding site in order to ultimatelysynthesize compounds capable of interacting with this site in such amanner that assembly of intact pili is prevented, inhibited or enhanced.

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27 874 base pairs nucleic acid single linear DNA (genomic) NO NOEscherichia coli CDS 1..720 sig_peptide 1..63 mat_peptide 64..717 M.Baga, M. Tennet, J.M. Normark, S.Norgren Mol. Biol. Rep. 12 169-178 19871 ATG ATT CGA AAA AAG ATT CTG ATG GCT GCC ATC CCC CTG TTT GTT ATA 48 MetIle Arg Lys Lys Ile Leu Met Ala Ala Ile Pro Leu Phe Val Ile -21 -20 -15-10 TCC GGG GCA GAC GCT GCT GTT TCG CTG GAC AGA ACC CGC GCG GTG TTT 96Ser Gly Ala Asp Ala Ala Val Ser Leu Asp Arg Thr Arg Ala Val Phe -5 1 510 GAC GGG AGT GAG AAG TCA ATG ACG CTT GAT ATC TCC AAT GAT AAC AAA 144Asp Gly Ser Glu Lys Ser Met Thr Leu Asp Ile Ser Asn Asp Asn Lys 15 20 25CAA CTG CCC TAT CTT GCT CAG GCA TGG ATA GAA AAT GAA AAT CAG GAA 192 GlnLeu Pro Tyr Leu Ala Gln Ala Trp Ile Glu Asn Glu Asn Gln Glu 30 35 40 AAAATT ATT ACA GGG CCG GTT ATT GCC ACC CCT CCG GTT CAG CGC CTT 240 Lys IleIle Thr Gly Pro Val Ile Ala Thr Pro Pro Val Gln Arg Leu 45 50 55 GAG CCGGGT GCG AAA AGC ATG GTC AGG CTG AGT ACC ACA CCG GAT ATC 288 Glu Pro GlyAla Lys Ser Met Val Arg Leu Ser Thr Thr Pro Asp Ile 60 65 70 75 AGT AAACTT CCT CAG GAC AGG GAA TCA CTG TTT TAT TTT AAT CTC AGG 336 Ser Lys LeuPro Gln Asp Arg Glu Ser Leu Phe Tyr Phe Asn Leu Arg 80 85 90 GAA ATA CCGCCG AGG AGT GAA AAG GCC AAT GTA CTG CAG ATA GCC TTA 384 Glu Ile Pro ProArg Ser Glu Lys Ala Asn Val Leu Gln Ile Ala Leu 95 100 105 CAG ACC AAAATA AAG CTT TTT TAT CGC CCG GCA GCA ATT AAA ACC AGA 432 Gln Thr Lys IleLys Leu Phe Tyr Arg Pro Ala Ala Ile Lys Thr Arg 110 115 120 CCA AAT GAAGTA TGG CAG GAC CAG TTA ATT CTG AAC AAA GTC AGC GGT 480 Pro Asn Glu ValTrp Gln Asp Gln Leu Ile Leu Asn Lys Val Ser Gly 125 130 135 GGG TAT CGTATT GAA AAC CCA ACG CCC TAT TAT GTC ACT GTT ATT GGT 528 Gly Tyr Arg IleGlu Asn Pro Thr Pro Tyr Tyr Val Thr Val Ile Gly 140 145 150 155 CTG GGAGGA AGT GAA AAG CAG GCA GAG GAA GGT GAG TTT GAA ACC GTG 576 Leu Gly GlySer Glu Lys Gln Ala Glu Glu Gly Glu Phe Glu Thr Val 160 165 170 ATG CTGTCT CCC CGT TCA GAG CAG ACA GTA AAA TCG GCA AAT TAT AAT 624 Met Leu SerPro Arg Ser Glu Gln Thr Val Lys Ser Ala Asn Tyr Asn 175 180 185 ACC CCTTAT CTG TCT TAT ATT AAT GAC TAT GGT GGT CGC CCG GTA CTG 672 Thr Pro TyrLeu Ser Tyr Ile Asn Asp Tyr Gly Gly Arg Pro Val Leu 190 195 200 TCG TTTATC TGT AAT GGT AGC CGT TGC TCT GTG AAA AAA GAG AAA TAA 720 Ser Phe IleCys Asn Gly Ser Arg Cys Ser Val Lys Lys Glu Lys * 205 210 215 TGTACCGCAATAACGGTTAA ATGCGGGTGG GATATTATGG TTGTGAATAA AACAACAGCA 780 GTACTGTATCTTATTGCACT GTCGCTGAGT GGTTTCATCC ATACTTTCCT GCGGGCTGAA 840 GAGCGGGGTATATACGATGA CGTCTTTACT GCAG 874 239 amino acids amino acid linear protein2 Met Ile Arg Lys Lys Ile Leu Met Ala Ala Ile Pro Leu Phe Val Ile -21-20 -15 -10 Ser Gly Ala Asp Ala Ala Val Ser Leu Asp Arg Thr Arg Ala ValPhe -5 1 5 10 Asp Gly Ser Glu Lys Ser Met Thr Leu Asp Ile Ser Asn AspAsn Lys 15 20 25 Gln Leu Pro Tyr Leu Ala Gln Ala Trp Ile Glu Asn Glu AsnGln Glu 30 35 40 Lys Ile Ile Thr Gly Pro Val Ile Ala Thr Pro Pro Val GlnArg Leu 45 50 55 Glu Pro Gly Ala Lys Ser Met Val Arg Leu Ser Thr Thr ProAsp Ile 60 65 70 75 Ser Lys Leu Pro Gln Asp Arg Glu Ser Leu Phe Tyr PheAsn Leu Arg 80 85 90 Glu Ile Pro Pro Arg Ser Glu Lys Ala Asn Val Leu GlnIle Ala Leu 95 100 105 Gln Thr Lys Ile Lys Leu Phe Tyr Arg Pro Ala AlaIle Lys Thr Arg 110 115 120 Pro Asn Glu Val Trp Gln Asp Gln Leu Ile LeuAsn Lys Val Ser Gly 125 130 135 Gly Tyr Arg Ile Glu Asn Pro Thr Pro TyrTyr Val Thr Val Ile Gly 140 145 150 155 Leu Gly Gly Ser Glu Lys Gln AlaGlu Glu Gly Glu Phe Glu Thr Val 160 165 170 Met Leu Ser Pro Arg Ser GluGln Thr Val Lys Ser Ala Asn Tyr Asn 175 180 185 Thr Pro Tyr Leu Ser TyrIle Asn Asp Tyr Gly Gly Arg Pro Val Leu 190 195 200 Ser Phe Ile Cys AsnGly Ser Arg Cys Ser Val Lys Lys Glu Lys 205 210 215 30 base pairsnucleic acid single linear DNA (genomic) 3 GTCAAACACC GCCGGAACTCGTCCAGGCGA 30 30 base pairs nucleic acid single linear DNA (genomic) 4CGGGCGATAA AAAAGAGCTA TTTTGGTCTG 30 27 base pairs nucleic acid singlelinear DNA (genomic) 5 GCGATAAAAA AGCATTATTT TCCTCTG 27 19 amino acidsamino acid single linear peptide 6 Gly Lys Arg Lys Pro Gly Glu Leu SerGly Ser Met Thr Met Val Leu 1 5 10 15 Ser Phe Pro 16 amino acids aminoacid single linear peptide 7 Lys Pro Gly Glu Leu Ser Gly Ser Met Thr MetVal Leu Ser Phe Pro 1 5 10 15 11 amino acids amino acid single linearpeptide 8 Ser Gly Ser Met Thr Met Val Leu Ser Phe Pro 1 5 10 7 aminoacids amino acid single linear peptide 9 Thr Met Val Leu Ser Phe Pro 1 519 amino acids amino acid single linear peptide 10 Gly Lys Arg Lys ProVal Glu Leu Ser Gly Ser Met Thr Met Val Leu 1 5 10 15 Ser Ser Pro 19amino acids amino acid single linear peptide 11 Gly Lys Arg Lys Pro GlyGlu Leu Ser Gly Ser Met Thr Met Val Leu 1 5 10 15 Ser Phe Pro 20 aminoacids amino acid single linear peptide 12 Glu Glu Gly Lys Arg Lys ProGly Glu Leu Ser Gly Ser Met Thr Met 1 5 10 15 Val Leu Ser Phe 20 19amino acids amino acid single linear peptide 13 Gln Asn Leu Ile Ala GlyPro Phe Ser Ala Thr Ala Thr Leu Val Ala 1 5 10 15 Ser Tyr Ser 19 aminoacids amino acid single linear peptide 14 Lys Lys Leu Glu Ala Gly AsnTyr Phe Ala Val Leu Gly Phe Arg Val 1 5 10 15 Asp Tyr Glu 19 amino acidsamino acid single linear peptide 15 Lys Ser Val Val Pro Gly Asp Tyr GluAla Thr Ala Thr Phe Glu Leu 1 5 10 15 Thr Tyr Arg 19 amino acids aminoacid single linear peptide 16 Gly Ile Leu Asn Gly Gly Asp Phe Gln ThrThr Ala Ser Met Ala Met 1 5 10 15 Ile Tyr Asn 16 amino acids amino acidsingle linear peptide 17 Tyr Ala Leu Ala Pro Asn Ala Val Ile Pro Thr SerLeu Ala Leu Leu 1 5 10 15 19 amino acids amino acid single linearpeptide 18 Lys Ser Val Val Pro Gly Asp Tyr Glu Ala Thr Ala Thr Phe GluLeu 1 5 10 15 Thr Tyr Arg 16 amino acids amino acid single linearpeptide 19 Leu Pro Ala Thr Asn Thr Leu Met Leu Ser Phe Asp Asn Val GlyGly 1 5 10 15 16 amino acids amino acid single linear peptide 20 Asp GlnIle Lys Gln Leu Pro Ala Thr Asn Thr Leu Met Leu Ser Phe 1 5 10 15 16amino acids amino acid single linear peptide 21 Tyr Lys Met Pro Tyr AspGln Ile Lys Gln Leu Pro Ala Thr Asn Thr 1 5 10 15 16 amino acids aminoacid single linear peptide 22 Gln His His Tyr Tyr Asp Leu Trp Gln AspHis Tyr Lys Met Pro Tyr 1 5 10 15 33 base pairs nucleic acid singlelinear DNA (genomic) 23 CCCCCCTGCA GATCAGATTA AGCAGCTACC TGC 33 34 basepairs nucleic acid single linear DNA (genomic) 24 CCCCTGCAGT AAAAATATCTCTGCTCAGAA ATAC 34 28 base pairs nucleic acid single linear DNA(genomic) 25 CCATCGATGA ACAGCCAGTC AGATAATC 28 61 base pairs nucleicacid single linear DNA (genomic) 26 GGAAAGAGAA AACCCGGGGA GCTATCTGGTTCTATGACTA TGGTTCTGAG TTTCCCCTGA 60 T 61 67 base pairs nucleic acidsingle linear DNA (genomic) 27 CGATCAGGGG AAACTCAGAA CCATAGTCATAGAACCAGAT AGCTCCCCGG GTTTTCTCTT 60 TCCTGCA 67

What is claimed is:
 1. A method for preventing, inhibiting or enhancing binding between at least one type of pilus subunit and at least one type of periplasmic molecular chaperone from the same species of pilus-forming bacteria, comprising providing a substance that interacts with the periplasmic molecular chaperone, wherein the periplasmic molecular chaperone binds pilus subunits during transport of the pilus subunits through the periplasmic space and/or during the process of assembly of the intact pilus, and introducing the substance into a cellular system comprising the pilus subunit and the periplasmic molecular chaperone in such a manner that binding of the pilus subunit to the periplasmic molecular chaperone is prevented, inhibited, or enhanced.
 2. A method according to claim 1, wherein the bacteria are selected from the group consisting of Haemophilus spp, Heliobacter spp, Pseudomonas aeruginosa, Mycoplasma spp, and all members of the Enterobacteriacieae family, including Escherischia spp, Salmonella spp, Bordetella spp, Yersinia spp, Klebsiella spp, and Proteus spp.
 3. A method according to claim 1, wherein the periplasmic molecular chaperone is a periplasmic protein selected from the group consisting of PapD, FimC, SfaE, FaeE, FanE, Cs3-1, F17D, C1pE, EcpD, Mrkb, FimB, SefB, HifB, MyfB, PsaB, PefD, YehC, MrpD, CssC, NfaE, AggD, and Caf1M.
 4. A method according to claim 1, wherein the prevention, inhibition or enhancement of the binding is accomplished by interaction with, in the periplasmic molecular chaperone, a binding site which is normally involved in binding to pilus subunits during transport of these pilus subunits through the periplasmic space and/or during the process of pilus assembly.
 5. A method according to claim 4, wherein the binding site is a binding site to which the carboxyl terminal part of a pilus subunit is capable of binding, and which comprises site points substantially identical to the invariant residues Arg-8 and Lys-112 in PapD, and a polypeptide fragment which is capable of interacting with a β-strand of the carboxyl terminal part of the pilus subunit, thereby stabilizing the binding of said subunit at the Arg-8 and Lys-112 site points of the binding site.
 6. A method according to claim 4, wherein the binding site is one to which the peptide G125′-140′ and/or the fusion peptide MBP-G1′-140′ and/or the peptide G1′-19′ is capable of binding.
 7. The method of claim 1 in which the substance is a compound of the general formula

wherein V₁ is O, S, SO, SO₂, CH₂, C(OH)H, CO or CS; W₁ is O, S, SO₂, SO₃, CH₂, or NH; R₁ is H; C₁₋₂₄ alkyl, C₁₋₂₄ alkenyl or C₁₋₂₄ alkynyl, which alkyl, alkenyl and alkynyl may be substituted with one or more substituents independently selected from OH, —CONH₂, —CSNH₂, —CONHOH, —CSNHOH, —NHCHO, —NHCONH₂, —NHCSNH₂, —NHSO₂NH₂ and —SO₂NH₂; acyl; or —(CH₂CH₂O)_(s)—H, wherein s=1, 2, 3; R₂ s a group of the formula

wherein A is —CH—(CH₂)_(n), or —CH═CH—(CH₂)_(n−1)— (n>20) or

B is —(CH₂)_(m)— or ═CH—(CH₂)_(m−1)— (m>0); X₂ is N, CH or C (when B is ═CH—(CH₂)_(m−1)—; and Y₂ is O, S, NH, H₂ or H (n=1); and 4>m+n>0, n<3, and m<3; or R₂ is a group of the formula

wherein A is —CH—(CH₂)_(n), or —CH═CH—(CH₂)_(n−1)— (n>0) or

B is —(CH₂)_(m)— or ═CH—(CH₂)_(m−1)— (m>0); and X′₂ is O, NH, CH₂ or S (when p=0); N or CH (when p=1); or C (when p=1 and B is═CH—(CH₂)_(m−1)—); V₂, Z_(2i), and W₂ are independently H, OH, —CONH₂, —CSNH₂, —CONOH, —CSNHOH, —NHCHO, —NHCONH₂, —NHCSNH₂, —NHSO₂NH₂, —SO₂NH₂, or V₂ and Z_(2i), or Z_(2i) and W₂ together form —NHC(O)NH—, —C(O)NHC(O)—, —NHS(O₂)NH—, —C(O)NHO—, —C(S)NHO—, —S(O₂)NHO—, or —S(O₂)NHC(O)—; 4>m+n>0, n<3, and m<3; or R₂ is a group —W₅—(C₁₋₅ alkyl or C₂₋₅ alkenyl or C₂₋₅ alkynyl) wherein W₅ is a bond or is selected from —O—, —S—, —So_(w)—, and NHC(O)—, and the C₁₋₅ alkyl or C₂₋₅ alkenyl or C₂₋₅ alkynyl moiety may be substituted with up to three groups selected independently from OH, —CONH₂, —CSNHOH, —NHCHO, —NHCONH₂, —NHCSNH₂, —NHSO₂NH₂, and —SO₂NH₂; —Z₁R₃ is —SO₂(OH), —PO(OH)₂, —OSO₂(OH), —NHSO₂(OH), —SPO(OH)₂, —CH₂COOH, tetrazol-5-yl or tetrazol-5-ylmethyl, or salts thereof; or Z₁ is —O—, —S—, —NH—, or —CH₂—, and R₃ is a group of the formula:

D is —CH₂—, —CO—, —SO₂—, —NH—SO₂—, —NH—CO, —O—PO(OH)— or a salt thereof; Z₃ is H, OH, —CONH₂, —CSNH₂, —CONHOH, —CSNHOH, —NHCHO, —NHCONH₂, —NHCSNH₂, —NHSO₂NH₂, —SO₂NH₂, —SO₂OH, —PO(OH)₂, —OSO₂(OH), —NHSO₂(OH), —COOH, tetrazolyl-5-yl or tetrazolyl-5-ylmethyl or a salt thereof, with the proviso that when D is —CH₂—, —CO—, —SO₂—, NHSO₂— or —NHCO—, then Z₃ is —SO₂(OH), —PO(OH)₂, —OSO₂(OH), —NHSO₂(OH), —COOH, tetrazolyl-5-yl or tetrazolyl-5-ylmethyl or a salt thereof; X₃ and Y₃ independently are H, NO₂, SO₂(NH₂), CONH₂, CF₃, or F; and U₄—W_(4r) is —CHCH—, —CH₂CH₂—, —C(OH)CH—, —CHC(OH)—, —CH(OH)CH₂—, CH₂CH(OH)—, —CH(OH)CH(OH)—, —C(O)NH—, —NHC(O)—; Y₁ is —O— or —S—; R₄ is H or, when Y₁ is S, S(CH₂)_(q)N(R₉)₃ ⁺ and q is an integer 2-4, where R₉ is H or CH₃; R₅ is H; C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, and the C₁₋₆ alkyl, C₂₋₆ alkenyl, or C₂₋₆ alkynyl moiety may be substituted with OH, —CONH₂, —CSNH₂, —CONHOH, —CSNHOH, —NHCHO, —NHCONH₂, —NHCSNH₂, —NHSO₂NH₂, or —SO₂NH₂; or aryl, aryl(C₁₋₂) alkyl, heterocyclyl, or heterocyclyl(C₁₋₂)alkyl which may optionally be substituted in the aryl or heterocyclyl moieties with one, two or three substituents selected independently from OH, F, Cl, NH₂, CONH₂, NHCOH, and SO₂NH₂; X₁ is —O—, —S— or NH; R₆ is H or, when X₁ is NH, acyl, HOCNH-Val-Met, HOCNH-Ile-(S,S)-dioxo-methionyl- or HOCNH-Val-(pyran-4-on-2-yl)-ala-nyl-; or a salt thereof.
 8. The method of claim 1 in which the substance is a pyranoside or salt thereof, which is selected from the group consisting of Ethyl 2,3-O-Dibenzoyl-4-O-benzyl-1-thio-β-D-glucohexopyranoside; Ethyl 6-O-acetyl-2,3-O-dibenzyol-4-O-benzyl-1-thio-β-D-glucohexopyranoside; Methylglycolyl 6-O-acetyl-2,3-O-dibenzyol-4-O-benzyl-β-D-glucohexopyranoside; 2-(Hydroxy)ethyl 4-O-benzyl-β-D-glucopyranoside; Sodium glycolyl 4-O-benzyl-β-D-glucohexopyranoside; Methyl 2-O-ethyl-4,6-O-(4′-methoxy)phenylmethylene-α-D-mannohexopyranoside; Methyl 2-O-ethyl-3-O-dimethyl-t-butylsilyl-4,6-O-(4′-methoxy)phenylmethylene-α-D-mannohexopyranoside; Methyl 2-O-ethyl-3-O-dimethyl-t-butylsilyl-4-O-(4′-methoxy)benzyl-α-D-mannohexopyranoside; methyl 2-O-ethyl-3-O-dimethyl-t-butylsilyl-6-O-(4′-methoxy)benzyl-α-D-mannohexopyranoside; Methyl 2-O-ethyl-3-O-dimethyl-t-butylsilyl-4-O-(4′-methoxy)benzyl-6(S)-phenyl-α-D-mannohexopyranoside; Methyl 2,3-anhydro-4,6-O-p-methoxybenzylidene-α-D-mannopyranoside; Methyl 3-azido-2-O-ethyl-4,6-O-p-methoxybenzylidene-α-D-altropyranoside; Methyl 3-azido-3-deoxy-2-O-ethyl-4-O-p-methoxybenzyl-α-D-altro-pyranosid; Methyl 3-azido-6-O-benzoyl-3-deoxy-2-O-ethyl-4-O-p-methoxybenzyl-α-D-altropyranoside; Methyl 6-O-benzoyl-3-deoxy-2-O-ethyl-4-O-p-methoxybenzyl-3-sulfamino-α-D-altropyranoside sodium salt; Methyl 6-O-benzoyl-3-deoxy-2-O-ethyl-3-sulfamino-α-D-altropyranoside ammonium salt; Methyl 3-azido-6-O-pivaloyl-3-deoxy-2-O-ethyl-4-O-p-methoxybenzyl-α-D-altropyranoside; Methyl 6-O-pivaloyl-3-deoxy-2-O-ethyl-4-O-p-methoxybenzyl-3-sulfamino-α-D-altropyranoside sodium salt; Methyl 6-O-pivaloyl-3-deoxy-2-O-ethyl-3-sulfamino-α-D-altropyranoside ammonium salt; Methyl 6-O-pivaloyl-3-deoxy-2-O-ethyl-4-O-p-methoxybenzyl-3-tbutyloxamido-α-D-altropyranoside; Methyl 6-O-pivaloyl-3-deoxy-2-O-ethyl-3-oxamido-α-D-altropyranoside ammonium salt; Methyl 3-azido-6-O-pyrrol-3′-ylcarboxyl-3-deoxy-2-O-ethyl-4-O-p-methoxybenzyl-α-D-altropyranoside; and Methyl 6-O-pyrrol-3′-ylcarboxyl-3-deoxy-2-O-ethyl-3-sulfamino-α-D-altropyranoside ammonium salt.
 9. The method of claim 1 in which the interaction is prevented or inhibited. 